Imidazopyridine compounds and uses thereof

ABSTRACT

This invention generally relates to substituted imidazopyridine compounds, particularly substituted 4-(imidazo[1,2-a]pyridin-2-yl)benzamide compounds and salts thereof. This invention also relates to pharmaceutical compositions and kits comprising such a compound, uses of such a compound (including, for example, treatment methods and medicament preparations), processes for making such a compound, and intermediates used in such processes.

CROSS-REFERENCE

This application is a continuation of U.S. patent application Ser. No.15/423,428, filed Feb. 2, 2017, which is a division of U.S. patentapplication Ser. No. 14/764,404, filed Jul. 29, 2015, now U.S. Pat. No.9,598,409, issued on Mar. 21, 2017, which is the National Stage Entry ofInternational Application No. PCT/CA2014/050062, filed Jan. 31, 2014,which claims the benefit of U.S. Provisional Patent Application No.61/759,123, filed Jan. 31, 2013, all of which are hereby incorporated byreference in their entirety.

FIELD OF THE INVENTION

This invention generally relates to substituted imidazopyridinecompounds, particularly substituted4-(imidazo[1,2-a]pyridin-2-yl)benzamide compounds and salts thereof.This invention also relates to pharmaceutical compositions and kitscomprising such a compound, uses of such a compound (including, forexample, treatment methods and medicament preparations), processes formaking such a compound, and intermediates used in such processes.

BACKGROUND

P2X purinoreceptors are a family of ion channels that are activated byextracellular adenosine triphosphate (ATP). Purinoreceptors have beenimplicated in a variety of biological functions, especially thoserelated to pain sensitivity. The P2X3 receptor subunit is a member ofthis family. It was originally cloned from rat dorsal root ganglia. Chenet al., Nature, vol. 377, pp. 428-431 (1995). The nucleotide and aminoacid sequences of both rat and human P2X3 are now known. Lewis, et al.,Nature, vol. 377, pp. 432-435 (1995); and Garcia-Guzman, et al., BrainRes. Mol. Brain Res., vol. 47, pp. 59-66 (1997).

P2X3 is reportedly involved in afferent pathways controlling urinarybladder volume reflexes. Consequently, inhibiting P2X3 may havetherapeutic potential for treating disorders of urine storage andvoiding, such as overactive bladder. Cockayne, et al., Nature, vol. 407,pp. 1011-1015 (2000).

P2X3 also is selectively expressed on nociceptive, small diametersensory neurons (i.e., neurons that are stimulated by pain or injury),which is consistent with a role in pain sensitivity. And blocking P2X3receptors has been reported to be analgesic in animal models of chronicinflammatory and neuropathic pain. Jarvis, et al., PNAS, 99, 17179-17184(2002). It is, therefore, believed that a method for reducing the P2X3level or activity would be useful for modulating pain sensation in asubject suffering from pain.

Various other disorders also have been discussed as being treatableusing compounds having P2X3 activity. See, e.g., WO2008/136756.

P2X3 also is capable of forming P2X2/3 heterodimers with P2X2, which isanother member of the P2X purinergic ligand-gated ion channel family.P2X2/3 is highly expressed on the terminals (central and peripheral) ofsensory neurons. Chen, et al., Nature, vol. 377, pp. 428-431 (1995).Results from recent studies also suggest that P2X2/3 is predominantlyexpressed (over P2X3) in bladder sensory neurons, and are likely to playa role in sensing of urinary bladder filling and nociception. Zhong, etal., Neuroscience, vol. 120, pp. 667-675 (2003).

In view of the foregoing, there is a need for new P2X3 and/or P2X2/3receptor ligands, particularly antagonists, that may be useful and safefor treating various disorders related to P2X3 and/or P2X2/3.

SUMMARY OF THE INVENTION

This invention comprises, inter alia, imidazopyridine compounds;treatment methods using the imidazopyridine compounds (e.g., use of theimidazopyridine to treat various disorders and as pharmacologicaltools); use of the imidazopyridine compounds to make medicaments;compositions comprising the imidazopyridine compounds (e.g.,pharmaceutical compositions); methods for manufacturing theimidazopyridine compounds; and intermediates used in such manufacturingmethods.

Briefly, this invention is directed, in part, to a compound of Formula Ior a salt thereof. Formula I corresponds to:

Here:

R¹ is selected from the group consisting of cyano, halogen, methyl, andethyl.

R² is selected from the group consisting of hydrogen, halogen, methyl,and ethyl.

R³ is selected from the group consisting of halogen, methyl, and ethyl.

R⁴ is selected from the group consisting of hydrogen, halogen, methyl,ethyl, and methoxy.

R⁵ and R⁶ are independently selected from the group consisting ofhydrogen, C₁-C₆-alkyl, and hydroxy-C₁-C₆-alkyl. Alternatively, R⁵ andR⁶, together with the nitrogen to which they are both attached, form a5- or 6-member heterocycloalkyl.

The heterocycloalkyl is optionally substituted with one or moresubstituents independently selected from the group consisting ofhalogen, hydroxyl, and C₁-C₄-alkyl.

R⁷ and R⁸ are independently selected from the group consisting ofhydrogen and C₁-C₄-alkyl.

R⁹ is selected from the group consisting of C₁-C₆-alkyl,C₃-C₆-cycloalkyl, C₁-C₆-alkyl-C₃-C₆-cycloalkyl, halo-C₁-C₆-alkyl,C₁-C₆-alkoxy, halo-C₁-C₆-alkoxy, and C₁-C₆-alkoxy-C₁-C₆-alkyl.

X is selected from a bond, CH₂, and O.

This invention also is directed, in part, to a pharmaceuticalcomposition that comprises a compound of Formula I or pharmaceuticallyacceptable salt thereof. In general, the composition also comprises atleast one pharmaceutically acceptable inert ingredient. Such inertingredients are sometimes collectively identified in this patent as“carriers, diluents, or excipients.” The composition may furthercomprise one or more additional active ingredients. For example, such acomposition may comprise one or more additional compounds of Formula Iand/or salts thereof. The composition also may, for example,alternatively or additionally comprise one or more active ingredientsother than a compound of Formula I or salt thereof.

This invention also is directed, in part, to a compound of Formula I ora pharmaceutically acceptable salt thereof for use as a medicament.

This invention also is directed, in part, to a kit comprising a compoundof Formula I or a pharmaceutically acceptable salt thereof.

This invention also is directed, in part, to the use of a compound ofFormula I or a pharmaceutically acceptable salt thereof formanufacturing a pharmaceutical composition (or “medicament”). Ingeneral, the composition also comprises at least one pharmaceuticallyacceptable carrier, diluent, or excipient. Such a composition mayfurther comprise one or more additional active ingredients. For example,such a composition may comprise one or more additional compounds ofFormula I and/or pharmaceutically acceptable salts thereof. Thecomposition also may, for example, alternatively or additionallycomprise one or more active ingredients other than a compound of FormulaI or salt thereof.

In some embodiments, the medicament is useful for treating a conditionassociated with P2X3 activity (particularly excessive activity) in ananimal (e.g., a human).

In some embodiments, the medicament is useful for treating a conditionassociated with P2X2/3 activity (particularly excessive activity) in ananimal (e.g., a human).

In some embodiments, the medicament is useful for treating pain in ananimal (e.g., a human).

In some embodiments, the medicament is useful for treating a urinarytract disorder in an animal (e.g., a human).

This invention also is directed, in part, to methods for treating adisorder in an animal (e.g., a human) in need of such treatment. Thesemethods comprise administering to the animal a compound of Formula I orpharmaceutically acceptable salt thereof. Such methods encompass theadministration of a compound of Formula I or pharmaceutically acceptablesalt thereof alone. They also encompass administering other ingredientsas well. For example, a compound of Formula I or pharmaceuticallyacceptable salt thereof will typically be administered as part of apharmaceutical composition that also comprises one or more carriers,diluents, or excipients. A compound of Formula I or pharmaceuticallyacceptable salt thereof also may be administered with one or moreadditional active ingredients. For example, one or more additionalcompounds of Formula I and/or pharmaceutically acceptable salts thereofmay be administered. Alternatively or additionally, one or more activeingredients other than a compound of Formula I or pharmaceuticallyacceptable salt thereof may be administered.

In some embodiments, the disorder comprises a disorder associated withP2X3 activity (particularly excessive activity).

In some embodiments, the disorder comprises a disorder associated withP2X2/3 activity (particularly excessive activity).

In some embodiments the disorder comprises pain.

In some embodiments, the disorder comprises a urinary tract disorder.

In general, when a compound of Formula I or salt thereof is administeredas the only active ingredient to treat a targeted disorder, theadministered amount of a compound of Formula I or pharmaceuticallyacceptable salt thereof is therapeutically effective to treat thetargeted disorder in the animal. When, in contrast, a compound ofFormula I or pharmaceutically acceptable salt thereof is administered incombination with one or more other active ingredients, the amount of acompound of Formula I or salt thereof and the amount(s) of the otheractive ingredient(s) are, together, therapeutically effective to treatthe targeted disorder in the mammal.

Further benefits of Applicants' invention will be apparent to oneskilled in the art from reading this specification.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Efficacy of Example 15 in rat FCA 96-hr model of inflammatorypain 30 minutes after p.o. dosing: Heat hyperalgesia (HH). Log freeCp=Logarithm of molar free drug concentration in plasma.

FIG. 2: Efficacy of Example 15 in rat FCA 96-hr model of inflammatorypain 30 minutes after p.o. dosing: mechanical hyperalgesia (MH). Logfree Cp=Logarithm of molar free drug concentration in plasma.

In the figures, the dotted curved line represents the 95% confidenceinterval of the best-fit curve. The dotted vertical line shows the invitro IC50 of the compound at the rat P2X3 assessed in FLIPR. The dottedhorizontal line shows the 50% reversal.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

This description of illustrative embodiments is intended only toacquaint others skilled in the art with Applicants' invention, itsprinciples, and its practical application so that others skilled in theart may adapt and apply the invention in its numerous forms, as they maybe best suited to the requirements of a particular use. This descriptionand its specific examples, while indicating embodiments of thisinvention, are intended for purposes of illustration only. Thisinvention, therefore, is not limited to the illustrative embodimentsdescribed in this specification, and may be variously modified.

As noted above, this invention is directed, in part, to a compound ofFormula I or a salt thereof. Formula I corresponds to:

Here:

R¹ is selected from the group consisting of cyano, halogen, methyl, andethyl.

In some embodiments, R¹ is chloro.

In some embodiments, R¹ is iodo.

In some embodiments, R¹ is fluoro.

In some embodiments, R¹ is bromo.

In some embodiments, R¹ is methyl.

In some embodiments, R¹ is ethyl.

In some embodiments, R¹ is cyano.

R² is selected from the group consisting of hydrogen, halogen, methyl,and ethyl.

In some embodiments, R² is hydrogen. In such embodiments, the compoundcorresponds in structure to Formula IA:

In some embodiments, R¹ is chloro, and R² is hydrogen.

In some embodiments, R¹ is iodo, and R² is hydrogen.

In some embodiments, R¹ is fluoro, and R² is hydrogen.

In some embodiments, R¹ is methyl, and R² is hydrogen.

In some embodiments, R¹ is cyano, and R² is hydrogen.

R³ is selected from the group consisting of halogen, methyl, and ethyl.

In some embodiments, R³ is fluoro.

In some embodiments, R³ is chloro.

In some embodiments, R³ is iodo.

In some embodiments, R³ is bromo.

In some embodiments, R³ is methyl.

In some embodiments, R³ is ethyl.

R⁴ is selected from the group consisting of hydrogen, halogen, methyl,ethyl, and methoxy.

In some embodiments, R⁴ is hydrogen. In such embodiments, the compoundcorresponds in structure to Formula IB:

In some embodiments, R⁴ is fluoro.

In some embodiments, R⁴ is chloro.

In some embodiments, R⁴ is iodo.

In some embodiments, R⁴ is bromo.

In some embodiments, R⁴ is methyl.

In some embodiments, R⁴ is ethyl.

In some embodiments, R⁴ is methoxy.

In some embodiments, R⁴ is selected from the group consisting ofhalogen, methyl, and ethyl. In some such embodiments, the compoundcorresponds in structure to Formula IC:

In other such embodiments, the compound corresponds in structure toFormula ID:

And, in still other embodiments, the compound corresponds in structureto Formula IE:

In some embodiments, R³ is fluoro, and R⁴ is hydrogen.

In some embodiments, R³ is chloro, and R⁴ is hydrogen.

In some embodiments, R³ is methyl, and R⁴ is hydrogen.

In some embodiments, R³ is fluoro, and R⁴ is fluoro.

In some embodiments, R³ is methyl, and R⁴ is fluoro.

In some embodiments, R³ is chloro, and R⁴ is chloro.

In some embodiments, R³ is methyl, and R⁴ is chloro.

In some embodiments, R³ is fluoro, and R⁴ is methyl.

In some embodiments, R³ is chloro, and R⁴ is methyl.

In some embodiments, R³ is methyl, and R⁴ is methyl.

In some embodiments, R⁵ and R⁶ are independently selected from the groupconsisting of hydrogen, C₁-C₆-alkyl, and hydroxy-C₁-C₆-alkyl.

In some embodiments, R⁵ is hydrogen such that the compound correspondsin structure to Formula IF:

In some embodiments, R⁵ is C₁-C₆-alkyl.

In some embodiments, R⁵ is methyl.

In some embodiments, R⁵ is ethyl.

In some embodiments, R⁵ is hydroxy-C₁-C₆-alkyl.

In some embodiments, R⁵ is 2-hydroxyethyl. Such a substituentcorresponds in structure to:

In some embodiments, R⁶ is hydrogen.

In some embodiments, R⁶ is C₁-C₆-alkyl.

In some embodiments, R⁶ is methyl.

In some embodiments, R⁶ is ethyl.

In some embodiments, R⁶ is hydroxy-C₁-C₆-alkyl.

In some embodiments, each of R⁵ and R⁶ is hydrogen.

In some embodiments, each of R⁵ and R⁶ is methyl.

In some embodiments, each of R⁵ and R⁶ is ethyl.

In some embodiments, R⁵ is hydrogen, and R⁶ is methyl.

In some embodiments, R⁵ is hydrogen, and R⁶ is ethyl.

In some embodiments, R⁵ is hydrogen, and R⁶ is 2-hydroxyethyl. In suchembodiments, the compound corresponds in structure to Formula IG:

In some embodiments, R⁵ is methyl, and R⁶ is ethyl.

In other embodiments, R⁵ and R⁶, together with the nitrogen to whichthey are both attached, form a 5- or 6-member heterocycloalkyl. Theheterocycloalkyl is optionally substituted with one or more substituentsindependently selected from the group consisting of halogen, hydroxyl,and C₁-C₄-alkyl. This heterocycloalkyl comprises a saturated,single-ring structure with 5 or 6 ring atoms that include at least 3carbon atoms; the nitrogen to which both R⁵ and R⁶ is attached; and,optionally, one additional heteroatom selected from the group consistingof nitrogen, sulfur, and oxygen. In some embodiments, theheterocycloalkyl is selected from the group consisting of pyrrolidinyl,piperidinyl, piperazinyl, morpholinyl, and thiomorpholinyl.

In some embodiments, R⁵ and R⁶, together with the nitrogen to which theyare both attached, form an unsubstituted 5- or 6-memberheterocycloalkyl.

In some embodiments, R⁵ and R⁶, together with the nitrogen to which theyare both attached, form unsubstituted pyrrolidinyl.

In some embodiments, R⁵ and R⁶, together with the nitrogen to which theyare both attached, form unsubstituted piperidinyl.

In some embodiments, R⁵ and R⁶, together with the nitrogen to which theyare both attached, form unsubstituted morpholinyl.

In some embodiments, R⁵ and R⁶, together with the nitrogen to which theyare both attached, form a 5- or 6-member heterocycloalkyl substitutedwith hydroxy.

In some embodiments, R⁵ and R⁶, together with the nitrogen to which theyare both attached, form hydroxylpyrrolidinyl.

R⁷ and R⁸ are independently selected from the group consisting ofhydrogen and C₁-C₄-alkyl.

In some embodiments, R⁷ is hydrogen.

In some embodiments, R⁷ is C₁-C₄-alkyl.

In some embodiments, R⁷ is methyl.

In some embodiments, R⁸ is hydrogen.

In some embodiments, R⁸ is C₁-C₄-alkyl.

In some embodiments, R⁸ is methyl.

In some embodiments, each of R⁷ and R⁸ is hydrogen. In such embodiments,the compound corresponds in structure to Formula IH:

In some embodiments, R⁷ is C₁-C₄-alkyl, and R⁸ is hydrogen. In some suchembodiments, the compound corresponds in structure to Formula II:

In other such embodiments, the compound corresponds in structure toFormula IJ:

In some embodiments, R⁷ is methyl, and R⁸ is hydrogen.

In some embodiments, each of R⁷ and R⁸ is C₁-C₄-alkyl. In some suchembodiments, R⁷ and R⁸ are bonded to the same carbon. For example, insome embodiments, the compound corresponds in structure to Formula IK:

In other embodiments, the compound corresponds in structure to FormulaIL:

In some embodiments, each of R⁷ and R⁸ is methyl.

R⁹ is selected from the group consisting of C₁-C₆-alkyl,C₃-C₆-cycloalkyl, C₁-C₆-alkyl-C₃-C₆-cycloalkyl, halo-C₁-C₆-alkyl,C₁-C₆-alkoxy, halo-C₁-C₆-alkoxy, and C₁-C₆-alkoxy-C₁-C₆-alkyl.

In some embodiments, R⁹ is C₁-C₆-alkyl.

In some embodiments, R⁹ is methyl.

In some embodiments, R⁹ is ethyl.

In some embodiments, R⁹ is isopropyl.

In some embodiments, R⁹ is halo-C₁-C₆-alkyl.

In some embodiments, R⁹ is monofluoroisopropyl.

In some embodiments, R⁹ is C₁-C₆-alkoxy.

In some embodiments, R⁹ is methoxy.

In some embodiments, R⁹ is t-butoxy.

In some embodiments, R⁹ is C₃-C₆-cycloalkyl.

In some embodiments, R⁹ is cyclopropyl.

In some embodiments, R⁹ is cyclobutyl.

In some embodiments, R⁹ is cyclobutyl.

In some embodiments, R⁹ is C₁-C₆-alkoxy-C₁-C₆-alkyl.

In some embodiments, R⁹ is methoxymethyl.

In some embodiments, R⁹ is C₁-C₆-alkyl-C₃-C₆-cycloalkyl. In some suchembodiments, for example, R⁹ is methylcylclopropyl.

X is selected from a bond, CH₂, and O.

In some embodiments, X is a bond. In such embodiments, the compoundcorresponds to Formula (IM):

In some such embodiments, for example, the compound corresponds toFormula (IN):

In some embodiments, X is CH₂. In such embodiments, the compoundcorresponds to Formula (IO):

In some such embodiments, for example, the compound corresponds toFormula (IP):

In other embodiments, the compound corresponds to Formula (IQ):

In some embodiments, X is O. In such embodiments, the compoundcorresponds to Formula (IR):

In some such embodiments, the compound corresponds in structure toFormula (IS):

In some embodiments of Formula (IS), the compound corresponds instructure to Formula (IT):

In other embodiments of Formula (IS), the compound corresponds instructure to Formula (IU):

In other embodiments of Formula (IS), the compound corresponds instructure to Formula (IV):

In other embodiments, the compound corresponds in structure to Formula(IW):

In some such embodiments for Formula (IW), the compound corresponds instructure to Formula (IX):

In other embodiments for Formula (IW), the compound corresponds instructure to Formula (IY):

Many of the compounds of this invention include at least one chiralcarbon, i.e., the carbon of the morpholinyl that is linked through amethylene group to the imidazopyridine. To the extent a structure inthis patent does not indicate the chirality, the structure is intendedto encompass any single chiral isomer corresponding to that structure,as well as any mixture of chiral isomers (e.g., the racemate). Thus, forexample, Formula I, which does not indicate the chirality, is intendedto encompass any single isomer corresponding to the structure, as wellas any mixture of chiral isomers. In some embodiments, a single chiralisomer is obtained by isolating it from a mixture of isomers (e.g., aracemate) using, for example, chiral chromatographic separation. Inother embodiments, a single chiral isomer is obtained through directsynthesis from, for example, a chiral starting material.

When a structure shows the chirality of a carbon, it depicts thedirection of one of the chiral carbon's substituents with a dark wedgeor hashed wedge. Unless otherwise indicated, the carbon substituentpointing in the opposite direction is hydrogen. This notation isconsistent with conventional organic chemistry nomenclature rules.

Contemplated salts of the compounds of this invention include both acidaddition salts and base addition salts. A salt may be advantageous dueto one or more of its chemical or physical properties, such as stabilityin differing temperatures and humidities, or a desirable solubility inwater, oil, or other solvent. In some instances, a salt may be used toaid in the isolation or purification of the compound. In someembodiments (particularly where the salt is intended for administrationto an animal, or is a reagent for use in making a compound or saltintended for administration to an animal), the salt is pharmaceuticallyacceptable.

In general, an acid addition salt can be prepared using variousinorganic or organic acids. Such salts can typically be formed by, forexample, mixing the compound with an acid (e.g., a stoichiometric amountof acid) using various methods known in the art. This mixing may occurin water, an organic solvent (e.g., ether, ethyl acetate, ethanol,isopropanol, or acetonitrile), or an aqueous/organic mixture. Examplesof inorganic acids that typically may be used to form acid additionsalts include hydrochloric, hydrobromic, hydroiodic, nitric, carbonic,sulfuric, and phosphoric acid. Examples of organic acids include, forexample, aliphatic, cycloaliphatic, aromatic, araliphatic, heterocyclic,carboxylic, and sulfonic classes of organic acids. Specific examples oforganic salts include cholate, sorbate, laurate, acetate,trifluoroacetate, formate, propionate, succinate, glycolate, gluconate,digluconate, lactate, malate, tartaric acid (and derivatives thereof,e.g., dibenzoyltartrate), citrate, ascorbate, glucuronate, maleate,fumarate, pyruvate, aspartate, glutamate, benzoate, anthranilic acid,mesylate, stearate, salicylate, p-hydroxybenzoate, phenylacetate,mandelate (and derivatives thereof), embonate (pamoate),ethanesulfonate, benzenesulfonate, pantothenate,2-hydroxyethanesulfonate, sulfanilate, cyclohexylaminosulfonate, algenicacid, β-hydroxybutyric acid, galactarate, galacturonate, adipate,alginate, butyrate, camphorate, camphorsulfonate,cyclopentanepropionate, dodecylsulfate, glycoheptanoate,glycerophosphate, heptanoate, hexanoate, nicotinate,2-naphthalesulfonate, oxalate, palmoate, pectinate, 3-phenylpropionate,picrate, pivalate, thiocyanate, tosylate, and undecanoate. In someembodiments, the salt comprises a hydrochloride, hydrobromide,phosphate, acetate, fumarate, maleate, tartrate, citrate,methanesulphonate, or p-toluenesulphonate salt.

With respect to base-addition salts, it may be possible to make analkali metal (such as sodium, potassium, or lithium) or an alkalineearth metal (such as a calcium) salt by treating a compound of thisinvention having a suitably acidic proton with an alkali metal oralkaline earth metal hydroxide or alkoxide (e.g., an ethoxide ormethoxide) or a suitably basic organic amine (e.g., a choline ormeglumine) in an aqueous medium.

The compounds of Formula I and salts thereof are intended to encompassany tautomer that may form. A “tautomer” is any other structural isomerthat exists in equilibrium resulting from the migration of a hydrogenatom, e.g., amide-imidic acid tautomerism.

It is contemplated that an amine of a compound of Formula I or a saltthereof may form an N-oxide. Such an N-oxide is intended to beencompassed by the compounds of Formula I and salts thereof. An N-oxidecan generally be formed by treating an amine with an oxidizing agent,such as hydrogen peroxide or a per-acid (e.g., a peroxycarboxylic acid).See, e.g., Advanced Organic Chemistry, by Jerry March, 4^(th) Edition,Wiley Interscience. N-oxides also can be made by reacting the amine withm-chloroperoxybenzoic acid (MCPBA), for example, in an inert solvent,such as dichloromethane. See L. W. Deady, Syn. Comm., 7, pp. 509-514(1977).

It is contemplated that a compound of Formula I or salt thereof couldform isolatable atropisomer in certain solvents at certain temperatures.The compounds of Formula I and salts thereof are intended to encompassany such atropisomers. Atropisomers can generally be isolated using, forexample, chiral LC.

The compounds of Formula I and salts thereof are intended to encompassany isotopically-labeled (or “radio-labeled”) derivatives of a compoundof Formula I or salt thereof. Such a derivative is a derivative of acompound of Formula I or salt thereof wherein one or more atoms arereplaced by an atom having an atomic mass or mass number different fromthe atomic mass or mass number typically found in nature. Examples ofradionuclides that may be incorporated include ²H (also written as “D”for deuterium), ³H (also written as “T” for tritium), ¹¹C, ¹³C, ¹⁴C,¹³N, ¹⁵N, ¹⁵O, ¹⁷O, ¹⁸O, ¹⁸F, ³⁶Cl, ⁸²Br, ⁷⁵Br, ⁷⁶Br, ⁷⁷Br, ¹²³I, ¹²⁴I,¹²⁵I, and ¹³¹I. The radionuclide that is used will depend on thespecific application of that radio-labeled derivative. For example, forin vitro receptor labeling and competition assays, ³H or ¹⁴C are oftenuseful. For radio-imaging applications, ¹¹C or ¹⁸F are often useful. Insome embodiments, the radionuclide is ³H. In some embodiments, theradionuclide is ¹⁴C. In some embodiments, the radionuclide is ¹¹C. Andin some embodiments, the radionuclide is ¹⁸F.

The compounds of Formula I and salts thereof are intended to cover allsolid state forms of the compounds of Formula I and salts thereof. Thecompounds of Formula I and salts thereof also are intended to encompassall solvated (e.g., hydrated) and unsolvated forms of the compounds ofFormula I and salts thereof.

The compounds of Formula I and salts thereof also are intended toencompass coupling partners in which a compound of Formula I or a saltthereof is linked to a coupling partner by, for example, beingchemically coupled to the compound or salt or physically associated withit. Examples of coupling partners include a label or reporter molecule,a supporting substrate, a carrier or transport molecule, an effector, adrug, an antibody, or an inhibitor. Coupling partners can be covalentlylinked to a compound of Formula I or salt thereof via an appropriatefunctional group on the compound, such as a hydroxyl, carboxyl, or aminogroup. Other derivatives include formulating a compound of Formula I ora salt thereof with liposomes.

This invention provides, in part, methods to treat various disorders inanimals, particularly mammals. Mammals include, for example, humans.Mammals also include, for example, companion animals (e.g., dogs, cats,and horses), livestock animals (e.g., cattle and swine); lab animals(e.g., mice and rats); and wild, zoo, and circus animals (e.g., bears,lions, tigers, apes, and monkeys).

As shown below in Example 48, compounds of this invention have beenobserved to modulate, and, in particular, act as antagonist against,P2X3. Accordingly, it is believed that the compounds and salts of thisinvention can be used to modulate P2X3 and/or P2X2/3 to treat variousconditions mediated by (or otherwise associated with) P2X3 and/orP2X2/3. In some embodiments, the compounds and salts of this inventionexhibit one or more of the following characteristics: desirable potency,desirable efficacy, desirable stability on the shelf, desirabletolerability for a range of patients, and desirable safety.

It is contemplated that a compound or salt of this invention may be usedto treat, for example, pain. Such pain may be, for example, chronicpain, neuropathic pain, acute pain, back pain, cancer pain, pain causedby rheumatoid arthritis, migraine, and visceral pain.

It also is contemplated that a compound or salt of this invention may beused to treat a urinary tract disorder. Such disorders include, forexample, over-active bladder (also known as urinary incontinence),pelvic hypersensitivity, and urethritis.

It also is contemplated that a compound or salt of this invention may beused to treat a gastrointestinal disorder. Such disorders include, forexample, constipation and functional gastrointestinal disorders (e.g.,irritable bowel syndrome or functional dyspepsia).

It also is contemplated that a compound or salt of this invention may beused to treat cancer.

It also is contemplated that a compound or salt of this invention may beused to treat a cardiovascular disorder or for cardioprotectionfollowing myocardial infarction.

It also is contemplated that a compound or salt of this invention may beuseful as an immunomodulator, especially for treating an autoimmunedisease (e.g., arthritis); for a skin graft, organ transplant, orsimilar surgical need; for a collagen disease; for an allergy; or as ananti-tumor or antiviral agent.

It also is contemplated that a compound or salt of this invention may beused to treat multiple sclerosis, Parkinson's disease, and Huntington'schorea.

It also is contemplated that a compound or salt of this invention may beuseful to treat depression, anxiety, a stress-related disorder (e.g., apost-traumatic stress disorder, panic disorder, social phobia, orobsessive compulsive disorder), premature ejaculation, a mental illness,traumatic brain injury, stroke, Alzheimer's disease, spinal injury, drugaddiction (e.g., treatment of alcohol, nicotine, opioid, or other drugabuse), or a disorder of the sympathetic nervous system (e.g.,hypertension).

It also is contemplated that a compound or salt of this invention may beused to treat diarrhea.

It also is contemplated that a compound or salt of this invention may beuseful to treat a pulmonary disorder, such as, for example, asthma, acough or lung edema.

It is contemplated that a compound of Formula I or a pharmaceuticallyacceptable salt thereof may be administered orally, buccally, vaginally,rectally, via inhalation, via insufflation, intranasally, sublingually,topically, or parenterally (e.g., intramuscularly, subcutaneously,intraperitoneally, intrathoracially, intravenously, epidurally,intrathecally, intracerebroventricularly, or by injection into thejoints).

In some embodiments, a compound or salt of this invention isadministered orally.

In some embodiments, a compound or salt of this invention isadministered intravenously.

In some embodiments, a compound or salt of this invention isadministered intramuscularly.

In some embodiments, a compound or salt of this invention is used tomake a medicament (i.e., a pharmaceutical composition). In general, thepharmaceutical composition comprises a therapeutically effective amountof the compound or salt. Pharmaceutical compositions comprising acompound or salt of this invention can vary widely. Although it iscontemplated that a compound or salt of this invention could beadministered by itself (i.e., without any other active or inactiveingredient), the pharmaceutical composition normally will insteadcomprise one or more additional active ingredients and/or inertingredients. The inert ingredients present in the pharmaceuticalcompositions of this invention are sometimes collectively referred to as“carriers, diluents, and excipients.” Methods for making pharmaceuticalcompositions and the use of carriers, diluents, and excipients are wellknown in the art. See, e.g., for example, Remington's PharmaceuticalSciences, Mack Publishing Company, Easton, Pa., 15th Edition, 1975.

Pharmaceutical compositions comprising a compound of Formula I orpharmaceutically acceptable salt thereof can vary widely. For example,it is contemplated that the compositions may be formulated for a varietyof suitable routes and means of administration, including oral, rectal,nasal, topical, buccal, sublingual, vaginal, inhalation, insufflation,or parenteral administration. It is contemplated that such compositionsmay, for example, be in the form of solids, aqueous or oily solutions,suspensions, emulsions, creams, ointments, mists, gels, nasal sprays,suppositories, finely divided powders, and aerosols or nebulisers forinhalation. In some embodiments, the composition comprises a solid orliquid dosage form that may be administered orally.

Solid form compositions may include, for example, powders, tablets,dispersible granules, capsules, cachets, and suppositories. A solidcarrier may comprise one or more substances. Such substances aregenerally inert. A carrier also may act as, for example, a diluent,flavoring agent, solubilizer, lubricant, preservative, stabilizer,suspending agent, binder, or disintegrating agent. It also may act as,for example, an encapsulating material. Examples of often suitablecarriers include pharmaceutical grade mannitol, lactose, magnesiumcarbonate, magnesium stearate, talc, lactose, sugar (e.g., glucose andsucrose), pectin, dextrin, starch, tragacanth, cellulose, cellulosederivatives (e.g., methyl cellulose and sodium carboxymethyl cellulose),sodium saccharin, low-melting wax, and cocoa butter.

In powders, the carrier is typically a finely divided solid, which is ina mixture with the finely divided active component. In tablets, theactive component is typically mixed with the carrier having thedesirable binding properties in suitable proportions and compacted intothe desired shape and size.

For preparing suppository compositions, a low-melting wax (e.g., amixture of fatty acid glycerides and cocoa butter) is typically firstmelted, followed by dispersing the active ingredient therein by, forexample, stirring. The molten homogeneous mixture is then poured intoconvenient-sized molds and allowed to cool and solidify. Examples ofnon-irritating excipients that may be present in suppositorycompositions include, for example, cocoa butter, glycerinated gelatin,hydrogenated vegetable oils, mixtures of polyethylene glycols of variousmolecular weights, and fatty acid esters of polyethylene glycol.

Liquid compositions can be prepared by, for example, dissolving ordispersing the compound or a salt of this invention in a carrier, suchas, for example, water, water/propylene glycol solutions, saline aqueousdextrose, glycerol, or ethanol. In some embodiments, aqueous solutionsfor oral administration can be prepared by dissolving a compound or saltof this invention in water with a solubilizer (e.g., a polyethyleneglycol). Colorants, flavoring agents, stabilizers, and thickeningagents, for example, also may be added. In some embodiments, aqueoussuspensions for oral use can be made by dispersing the compound or saltof this invention in a finely divided form in water, together with aviscous material, such as, for example, one or more natural syntheticgums, resins, methyl cellulose, sodium carboxymethyl cellulose, or othersuspending agents. If desired, the liquid composition also may containother non-toxic auxiliary inert ingredients, such as, for example,wetting or emulsifying agents, pH buffering agents and the like, forexample, sodium acetate, sorbitan monolaurate, triethanolamine sodiumacetate, sorbitan monolaurate, triethanolamine oleate, etc. Suchcompositions also may contain other ingredients, such as, for example,one or more pharmaceutical adjuvants.

In some embodiments, the pharmaceutical composition comprises from about0.05% to about 99% (by weight) of a compound or salt of this invention.In some such embodiments, for example, the pharmaceutical compositioncomprises from about 0.10% to about 50% (by weight) of a compound orsalt of this invention.

When a compound or salt of this invention is administered as a soletherapy for treating a disorder, a “therapeutically effective amount” isan amount sufficient to reduce or completely alleviate symptoms or otherdetrimental effects of the disorder; cure the disorder; reverse,completely stop, or slow the progress of the disorder; reduce the riskof the disorder getting worse; or delay or reduce the risk of onset ofthe disorder.

The optimum dosage and frequency of administration will depend on theparticular condition being treated and its severity; the species of thepatient; the age, size and weight, diet, and general physical conditionof the particular patient; brain/body weight ratio; other medication thepatient may be taking; the route of administration; the formulation; andvarious other factors known to physicians (in the context of humanpatients), veterinarians (in the context of non-human patients), andothers skilled in the art.

It is contemplated that in some embodiments, the optimum amount of acompound or salt of this invention is at least about 10 pg/kg of bodyweight per day. In some embodiments, the optimum amount is no greaterthan about 100 mg/kg of body weight per day. In some embodiments, theoptimum amount is from about 10 pg/kg to about 100 mg/kg of body weightper day. In some embodiments, the optimum amount is from about 0.01 toabout 10 mg/kg of body weight per day. In some embodiments, the optimumamount is from about 2 to about 20 mg/kg of body weight per day. In someembodiments, the optimum amount is from about 2.5 to about 8 mg/kg ofbody weight per day. In still other embodiments, the optimum amount isfrom about 0.8 to about 2.5 mg/kg of body weight per day.

It is contemplated that the pharmaceutical compositions can be in one ormore unit dosage forms. Accordingly, the composition may be divided intounit doses containing appropriate quantities of the active component.The unit dosage form can be, for example, a capsule, cachet, or tabletitself, or it can be the appropriate number of any of these in packagedforms. The unit dosage form alternatively can be a packaged preparationin which the package contains discrete quantities of the composition,such as, for example, packeted tablets, capsules, or powders in vials orampoules. Unit dosage forms may be prepared by, for example, variousmethods well known in the art of pharmacy.

It is contemplated that a dosage can be given once daily or in divideddoses, such as, for example, from 2 to 4 times per day. In someembodiments, the dose is conventionally formulated in an oral dosageform by compounding from about 5 to about 250 mg per unit of dosagewith, for example, one or more inert or active ingredients usingaccepted pharmaceutical practices.

In some embodiments, a compound or salt of this invention isadministered concurrently, simultaneously, sequentially, or separatelywith one or more other pharmaceutically active compounds. In some suchembodiments, the other pharmaceutically active compound(s) is/areselected from the following:

-   (i) Antidepressants, which are contemplated to include, for example,    one or more of agomelatine, amitriptyline, amoxapine, bupropion,    citalopram, clomipramine, desipramine, doxepin duloxetine,    elzasonan, escitalopram, fluvoxamine, fluoxetine, gepirone,    imipramine, ipsapirone, maprotiline, mirtazeprine, nortriptyline,    nefazodone, paroxetine, phenelzine, protriptyline, ramelteon,    reboxetine, robalzotan, selegiline, sertraline, sibutramine,    thionisoxetine, tranylcypromaine, trazodone, trimipramine,    venlafaxine and equivalents and pharmaceutically active isomer(s)    and metabolite(s) thereof.-   (ii) Antipsychotics, which are contemplated to include, for example,    one or more of quetiapine and pharmaceutically active isomer(s) and    metabolite(s) thereof; and amisulpride, aripiprazole, asenapine,    benzisoxidil, bifeprunox, carbamazepine, clozapine, chlorpromazine,    debenzapine, dibenzapine, divalproex, droperidol, duloxetine,    eszopiclone, fluphenazine, haloperidol, iloperidone, lamotrigine,    lithium, loxapine, mesoridazine, molindone, olanzapine,    paliperidone, perlapine, perphenazine, phenothiazine,    phenylbutylpiperidine, pimozide, prochlorperazine, risperidone,    sertindole, sulpiride, suproclone, suriclone, thioridazine,    thiothixene, trifluoperazine, trimetozine, valproate, valproic acid,    zopiclone, zotepine, ziprasidone, and equivalents thereof.-   (iii) Anxiolytics, which are contemplated to include, for example,    one or more of alnespirone, azapirones, benzodiazepines,    barbiturates such as adinazolam, alprazolam, balezepam, bentazepam,    bromazepam, brotizolam, buspirone, clonazepam, clorazepate,    chlordiazepoxide, cyprazepam, diazepam, diphenhydramine, estazolam,    fenobam, flunitrazepam, flurazepam, fosazepam, lorazepam,    lormetazepam, meprobamate, midazolam, nitrazepam, oxazepam,    prazepam, quazepam, reclazepam, suriclone, tracazolate, trepipam,    temazepam, triazolam, uldazepam, zolazepam and equivalents and    pharmaceutically active isomer(s) and metabolite(s) thereof.-   (iv) Anticonvulsants, which are contemplated to include, for    example, one or more of carbamazepine, oxcarbazepine, valproate,    lamotrogine, gabapentin, topiramate, phenytoin, ethoxuximide, and    equivalents and pharmaceutically active isomer(s) and metabolite(s)    thereof.-   (v) Alzheimer's therapies, which are contemplated to include, for    example, donepezil, galantamine, memantine, rivastigmine, tacrine    and equivalents and pharmaceutically active isomer(s) and    metabolite(s) thereof.-   (vi) Parkinson's therapies and agents for the treatment of    extrapyramidal symptoms, which are contemplated to include, for    example, one or more of levodopa, carbidopa, amantadine,    pramipexole, ropinirole, pergolide, cabergoline, apomorphine,    bromocriptine, MAOB inhibitors (e.g., selegine and rasagiline), COMT    inhibitors (e.g., entacapone and tolcapone), alpha-2 inhibitors,    anticholinergics (e.g., benztropine, biperiden, orphenadrine,    procyclidine, and trihexyphenidyl), dopamine reuptake inhibitors,    NMDA antagonists, Nicotine agonists, Dopamine agonists, and    inhibitors of neuronal nitric oxide synthase, and equivalents and    pharmaceutically active isomer(s) and metabolite(s) thereof.-   (vii) Stroke therapies, which are contemplated to include, for    example, one or more of abciximab, activase, disufenton sodium,    citicoline, crobenetine, desmoteplase, repinotan, traxoprodil, and    equivalents and pharmaceutically active isomer(s) and metabolite(s)    thereof.-   (viii) Urinary incontinence therapies, which are contemplated to    include, for example, one or more of darafenacin, dicyclomine,    falvoxate, imipramine, desipramine, oxybutynin, propiverine,    propanthedine, robalzotan, solifenacin, alfazosin, doxazosin,    terazosin, tolterodine, and equivalents and pharmaceutically active    isomer(s) and metabolite(s) thereof.-   (ix) Insomnia therapies, which are contemplated to include, for    example, one or more of allobarbital, alonimid, amobarbital,    benzoctamine, butabarbital, capuride, chloral, cloperidone,    clorethate, dexclamol, estazolam, eszopicline, ethchlorvynol,    etomidate, flurazepam, glutethimide, halazepam, hydroxyzine,    mecloqualone, melatonin, mephobarbital, methaqualone, midaflur,    midazolam, nisobamate, pagoclone, pentobarbital, perlapine,    phenobarbital, propofol, quazepam, ramelteon, roletamide,    suproclone, temazepam, triazolam, triclofos, secobarbital, zaleplon,    zolpidem, zopiclone, and equivalents and pharmaceutically active    isomer(s) and metabolite(s) thereof.-   (x) Mood stabilizers, which are contemplated to include, for    example, one or more of carbamazepine, divalproex, gabapentin,    lamotrigine, lithium, olanzapine, quetiapine, valproate, valproic    acid, verapamil, and equivalents and pharmaceutically active    isomer(s) and metabolite(s) thereof.-   (xi) Medications for treating obesity, such as, for example,    orlistat, sibutramine, rimonabant, and equivalents and    pharmaceutically active isomer(s) and metabolite(s) thereof.-   (xii) Agents for treating ADHD, which are contemplated to include,    for example, one or more of amphetamine, methamphetamine,    dextroamphetamine, atomoxetine, methylphenidate, dexmethylphenidate,    modafinil, and equivalents and pharmaceutically active isomer(s) and    metabolite(s) thereof.-   (xiii) Agents used to treat substance abuse disorders, dependence,    and withdrawal, which are contemplated to include, for example, one    or more of nicotine replacement therapies (e.g., gum, patches, and    nasal spray); nicotinergic receptor agonists, partial agonists, and    antagonists, (e.g., varenicline); acomprosate; bupropion; clonidine;    disulfiram; methadone; naloxone; naltrexone; and equivalents and    pharmaceutically active isomer(s) and metabolite(s) thereof.

In some embodiments, the other pharmaceutically active ingredient(s)comprises an atypical antipsychotic agent. Atypical antipsychotic agentsinclude, for example, olanzapine (marketed as Zyprexa), aripiprazole(marketed as Abilify), risperidone (marketed as Risperdal), quetiapine(marketed as Seroquel), clozapine (marketed as Clozaril), ziprasidone(marketed as Geodon), and olanzapine/fluoxetine (marketed as Symbyax).

In some embodiments, the other pharmaceutically active ingredient(s)comprises a selective serotonin reuptake inhibitor (or“serotonin-specific reuptake inhibitor” or SSRI”). Such agents include,for example, fluoxetine (marketed as, for example, Prozac), paroxetine(marketed as, for example, Paxil), citalopram (marketed as, for example,Celexa), dapoxetine, mesembrine, excitalopram (marketed as, for example,Lexapro), fluvoxamine (marketed as, for examle, Luvox), zimelidine(marketed as, for example, Zelmid), and sertraline (marketed as, forexample, Zoloft).

In some embodiments, a compound or salt of this invention isadministered as part of a combination therapy with radiotherapy.

In some embodiments, a compound or salt of this invention isadministered as a combination therapy with chemotherapy. Suchchemotherapy may include one or more of the following categories ofanti-tumour agents:

-   (i) Antiproliferative/antineoplastic drugs, which are contemplated    to include, for example, alkylating agents, such as cis-platin,    oxaliplatin, carboplatin, cyclophosphamide, nitrogen mustard,    melphalan, chlorambucil, busulphan, temozolamide, and nitrosoureas;    antimetabolites, such as gemcitabine and antifolates (e.g.,    fluoropyrimidines (like 5-fluorouracil and tegafur), raltitrexed,    methotrexate, cytosine arabinoside, and hydroxyurea); antitumour    antibiotics, such as anthracyclines (e.g., adriamycin, bleomycin,    doxorubicin, daunomycin, epirubicin, idarubicin, mitomycin-C,    dactinomycin and mithramycin); antimitotic agents, such as vinca    alkaloids (e.g., vincristine, vinblastine, vindesine, and    vinorelbine), taxoids (e.g., taxol and taxotere), and polokinase    inhibitors; and topoisomerase inhibitors, such as    epipodophyllotoxins (e.g., etoposide and teniposide), amsacrine,    topotecan, and camptothecin.-   (ii) Cytostatic agents, which are contemplated to include, for    example, antioestrogens, such as tamoxifen, fulvestrant, toremifene,    raloxifene, droloxifene, and iodoxyfene; antiandrogens, such as    bicalutamide, flutamide, nilutamide, and cyproterone acetate; LHRH    antagonists; LHRH agonists, such as goserelin, leuprorelin, and    buserelin; progestogens, such as megestrol acetate; aromatase    inhibitors, such as anastrozole, letrozole, vorazole, and    exemestane; and 5α-reductase inhibitors, such as finasteride.-   (iii) Anti-invasion agents, which are contemplated to include, for    example, c-Src kinase family inhibitors, such as    4-(6-chloro-2,3-methylenedioxyanilino)-7-[2-(4-methylpiperazin-1-yl)ethoxy]-5-tetrahydropyran-4-yloxyquinazoline    (AZD0530, Int'l Patent Appl. Publ. WOO1/94341),    N-(2-chloro-6-methylphenyl)-2-{6-[4-(2-hydroxyethyl)piperazin-1-yl]-2-methylpyrimidin-4-ylamino}thiazole-5-carboxamide    (dasatinib, BMS-354825, J. Med. Chem., vol. 47, pp. 6658-6661    (2004)), and bosutinib (SKI-606); metalloproteinase inhibitors, such    as marimastat; inhibitors of urokinase plasminogen activator    receptor function; and antibodies to heparanase.-   (iv) Inhibitors of growth factor function, which are contemplated to    include, for example, growth factor antibodies; growth factor    receptor antibodies, such as the anti-erbB2 antibody trastuzumab    (Herceptin™), the anti-EGFR antibody panitumumab, the anti-erbB1    antibody cetuximab (Erbitux, C225), and growth factor or growth    factor receptor antibodies disclosed by Stern et al., Critical    reviews in oncology/haematology, vol. 54, pp. 11-29 (2005); tyrosine    kinase inhibitors, such as inhibitors of the epidermal growth factor    family (e.g., EGFR family tyrosine kinase inhibitors like    N-(3-chloro-4-fluorophenyl)-7-methoxy-6-(3-morpholinopropoxy)quinazolin-4-amine    (gefitinib, ZD1839),    N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)quinazolin-4-amine    (erlotinib, OSI-774), and    6-acrylamido-N-(3-chloro-4-fluorophenyl)-7-(3-morpholinopropoxy)-quinazolin-4-amine    (CI 1033)) and erbB2 tyrosine kinase inhibitors (e.g., lapatinib);    inhibitors of the hepatocyte growth factor family; inhibitors of the    insulin growth factor family; inhibitors of the platelet-derived    growth factor family, such as imatinib and nilotinib (AMN107);    inhibitors of serine/threonine kinases, such as Ras/Raf signalling    inhibitors (e.g., farnesyl transferase inhibitors like sorafenib    (BAY 43-9006), tipifarnib (R115777), and lonafarnib (SCH66336));    inhibitors of cell signalling through MEK and/or AKT kinases; c-kit    inhibitors; abl kinase inhibitors, PI3 kinase inhibitors; Plt3    kinase inhibitors; CSF-1R kinase inhibitors; IGF receptor    (insulin-like growth factor) kinase inhibitors); aurora kinase    inhibitors, such as AZD1152, PH739358, VX-680, MLN8054, R763, MP235,    MP529, VX-528, and AX39459; and cyclin dependent kinase inhibitors,    such as CDK2 and CDK4 inhibitors.-   (v) Antiangiogenic agents, which are contemplated to include, for    example, those that inhibit the effects of vascular endothelial    growth factor, such as anti-vascular endothelial cell growth factor    antibody bevacizumab (Avastin™) and a VEGF receptor tyrosine kinase    inhibitor (e.g., vandetanib (ZD6474), vatalanib (PTK787), sunitinib    (SU11248), axitinib (AG-013736), pazopanib (GW 786034), and    4-(4-fluoro-2-methylindol-5-yloxy)-6-methoxy-7-(3-pyrrolidin-1-ylpropoxy)quinazoline    (AZD2171, Example 240 in Intl. Patent Appl. Publ. WO 00/47212);    compounds disclosed in Int'l Patent Appl. Publ. WO97/22596, WO    97/30035, WO 97/32856, and WO 98/13354; and compounds that work by    other mechanisms, such as linomide, inhibitors of integrin αvβ3    function, and angiostatin.-   (vi) Vascular damaging agents, which are contemplated to include,    for example, combretastatin A4 and compounds disclosed in Int'l    Patent Appl. Publ. WO 99/02166, WO 00/40529, WO 00/41669, WO    01/92224, WO 02/04434, and WO 02/08213.-   (vii) Endothelin receptor antagonists, which are contemplated to    include, for example, zibotentan (ZD4054) and atrasentan.-   (viii) Antisense therapies, which are contemplated to include, for    example, those that are directed to the targets listed above, such    as ISIS 2503 (an anti-ras antisense).-   (ix) Gene therapy approaches, which are contemplated to include, for    example, approaches to replace aberrant genes, such as aberrant p53,    BRCA1, or BRCA2; GDEPT (gene-directed enzyme pro-drug therapy)    approaches, such as those using cytosine deaminase, thymidine    kinase, or a bacterial nitroreductase enzyme; and approaches to    increase patient tolerance to chemotherapy or radiotherapy, such as    multi-drug resistance gene therapy.-   (x) Immunotherapy approaches, which are contemplated to include, for    example, ex-vivo and in-vivo approaches to increase the    immunogenicity of patient tumour cells, such as transfection with    cytokines (e.g., interleukin 2, interleukin 4, or    granulocyte-macrophage colony stimulating factor); approaches to    decrease T-cell anergy; approaches using transfected immune cells,    such as cytokine-transfected dendritic cells; approaches using    cytokine-transfected tumour cell lines; and approaches using    anti-idiotypic antibodies.

It also is contemplated that a compound or salt of this invention may beuseful as an analgesic agent for use during general anesthesia ormonitored anesthesia care. Combinations of agents with differentproperties are often used to achieve a balance of effects needed tomaintain the anesthetic state (e.g., amnesia, analgesia, musclerelaxation, and sedation). Such a combination may include, for example,one or more inhaled anesthetics, hypnotics, anxiolytics, neuromuscularblockers, and/or opioids.

In some embodiments in which a combination therapy is used, the amountof the compound or salt of this invention and the amount of the otherpharmaceutically active agent(s) are, when combined, therapeuticallyeffective to treat a targeted disorder in the animal patient. In thiscontext, the combined amounts are “therapeutically effective amount” ifthey are, when combined, sufficient to reduce or completely alleviatesymptoms or other detrimental effects of the disorder; cure thedisorder; reverse, completely stop, or slow the progress of thedisorder; reduce the risk of the disorder getting worse; or delay orreduce the risk of onset of the disorder. Typically, such amounts may bedetermined by one skilled in the art by, for example, starting with thedosage range described in this patent for the compound or salt of thisinvention and an approved or otherwise published dosage range(s) of theother pharmaceutically active compound(s).

When used in a combination therapy, it is contemplated that the compoundor salt of this invention and the other active ingredients may beadministered in a single composition, completely separate compositions,or a combination thereof. It also is contemplated that the activeingredients may be administered concurrently, simultaneously,sequentially, or separately. The particular composition(s) and dosingfrequency(ies) of the combination therapy will depend on a variety offactors, including, for example, the route of administration, thecondition being treated, the species of the patient, any potentialinteractions between the active ingredients when combined into a singlecomposition, any interactions between the active ingredients when theyare administered to the animal patient, and various other factors knownto physicians (in the context of human patients), veterinarians (in thecontext of non-human patients), and others skilled in the art.

This invention also is directed, in part, to a kit comprising a compoundof Formula I or a salt thereof. In some embodiments, the kit furthercomprises one or more additional components, such as, for example: (a)an apparatus for administering the compound of Formula I or a saltthereof; (b) instructions for administering the compound of Formula I ora salt thereof; (c) a carrier, diluent, or excipient (e.g., are-suspending agent); and (d) an additional active ingredient, which maybe in the same and/or different dosage forms as the compound of FormulaI or salt thereof. In some embodiments (particularly when the kit isintended for use in administering the compound of Formula I or saltthereof to an animal patient), the salt is a pharmaceutically acceptablesalt.

EXAMPLES

The following examples are merely illustrative of embodiments of theinvention, and not limiting to the remainder of this disclosure in anyway.

In some instances in the following examples, compound structures areassociated with compound names. In general, such names were generatedfrom the structures using AutoNom 2000 within ISIS/Draw, ChemDraw 9.0.7,ISIS/Draw 2.5SP4, or ChemDraw 11.0.2. AutoNom (Automatic Nomenclature)and ChemDraw contain programs that assign systematic IUPAC(International Union of Pure and Applied Chemistry) chemical names todrawn structures at the press of a button. In some instances, however,the chemical names were manually revised to ensure compliance with IUPACnaming conventions. If there are any differences between a structure andname for a compound, the compound should be identified by the structureunless the context indicates otherwise.

Compound Preparation

Examples 1-47 below illustrate the preparation of a variety of differentcompounds of this invention and intermediates for making such compounds.It is expected that one skilled in the art of organic synthesis, afterreading these examples alone or in combination with the generalknowledge in the art, can adapt and apply the methods to make anycompound encompassed by this invention. The general knowledge in the artincludes, for example:

-   A) Conventional procedures for using protective groups and examples    of suitable protective groups, which are described in, for example,    Protective Groups in Organic Synthesis, T. W. Green, P. G. M. Wuts,    Wiley-Interscience, New York (1999).-   B) References discussing various organic synthesis reactions,    include textbooks of organic chemistry, such as, for example,    Advanced Organic Chemistry, March 4th ed, McGraw Hill (1992); and    Organic Synthesis, Smith, McGraw Hill, (1994). They also include,    for example, R. C. Larock, Comprehensive Organic Transformations,    2nd ed., Wiley-VCH: New York (1999); F. A. Carey; R. J. Sundberg,    Advanced Organic Chemistry, 2nd ed., Plenum Press: New York    (1984); L. S. Hegedus, Transition Metals in the Synthesis of Complex    Organic Molecules, 2nd ed., University Science Books: Mill Valley, C    A (1994); L. A. Paquette, Ed., The Encyclopedia of Reagents for    Organic Synthesis, John Wiley: New York (1994); A. R. Katritzky, O.    Meth-Cohn, C W. Rees, Eds., Comprehensive Organic Functional Group    Transformations, Pergamon Press: Oxford, UK (1995); G. Wilkinson; F.    G A. Stone; E. W. Abel, Eds., Comprehensive Organometallic    Chemistry, Pergamon Press: Oxford, UK (1982); B. M. Trost; I.    Fleming, Comprehensive Organic Synthesis, Pergamon Press: Oxford, UK    (1991); A. R. Katritzky, C W. Rees Eds., Comprehensive Heterocyclic    Chemistry, Pergamon Press: Oxford, UK (1984); A. R. Katritzky; CW.    Rees, E. F. V. Scriven, Eds., Comprehensive Heterocyclic Chemistry    II, Pergamon Press: Oxford, UK (1996); C. Hansen; P. G.    Sammes; J. B. Taylor, Eds., Comprehensive Medicinal Chemistry:    Pergamon Press: Oxford, UK (1990). In addition, recurring reviews of    synthetic methodology and related topics include: Organic Reactions,    John Wiley: New York; Organic Syntheses; John Wiley: New York; The    Total Synthesis of Natural Products, John Wiley: New York; The    Organic Chemistry of Drug Synthesis, John Wiley: New York; Annual    Reports in Organic Synthesis, Academic Press: San Diego Calif.; and    Methoden der Organischen Chemie (Houben-Weyl), Thieme: Stuttgart,    Germany.-   C) References discussing heterocyclic chemistry include, for    example, example, Heterocyclic Chemistry, J. A. Joule, K.    Mills, G. F. Smith, 3rd ed., Cheapman and Hall, p. 189-225 (1995);    and Heterocyclic Chemistry, T. L. Gilchrist, 2^(nd) ed. Longman    Scientific and Technical, p. 248-282 (1992).-   D) Databases of synthetic transformations, including Chemical    Abstracts, which may be searched using either CAS Online or    SciFinder; and Handbuch der Organischen Chemie (Beilstein), which    may be searched using SpotFire.

All starting materials in the following compound preparation examplesare commercially available or described in the literature. Air andmoisture-sensitive liquids and solutions were transferred via syringe orcannula, and introduced into reaction vessels through rubber septa.Commercial grade reagents and solvents were used without furtherpurification. The terms “concentration under reduced pressure” and“evaporated under reduce pressure” or “concentrated in vacuo” refer touse of a Buchi rotary evaporator at approximately 15 mm of Hg.

Microwave heating was performed either on a CEM Discover LabMate or on aBiotage Initiator System at the indicated temperature in the recommendedmicrowave tubes.

Column chromatography (flash chromatography) was performed using 32-63micron, 60 Å, silica gel prepacked cartridges (on a Biotage or ISCOsystem), or a glass column and air pressure. Preparative HPLC or LCMS(high pH or low pH) was performed using, for example, a Waters X-bridgePrep C₁₈ OBD (column size: 30×50 mm; particle size: 5 μm; mobile phaseA: water 10 mM NH₄HCO₃ (pH 10) or water with 0.1% TFA; and mobile phaseB: MeCN). Supercritical-fluid chromatography (SFC) was performed using aMiniGram SFC instrument from Mettler Toledo with a normal-phaseChiralCel OD-H or OJ-H column or a ChiralPak AS-H or AD-H or IC column(column size: 10×250 mm; particle size: 5 mm; flow rate: 10 mL/min) orChiralPak IA or Lux Cellulose-2 or Lux Amylose-2 (column size: 4.6×250mm; particle size: 5 mm; flow rate: 3.5 mL/min); Eluent: CO₂, with MeOHor i-PrOH or EtOH+0.1% dimethylethylamine (DMEA) or 1:1isopropanol:acetonitrile+0.1% DMEA as a modifier; temperature: 35° C.;back pressure: 100 Bar; and UV detection at wavelength 215-254 nm.

Mass spectra were recorder using either Single-Quad mass spectrometersequipped with an electrospray ion source (ES) operated in a positive ornegative ion mode or a Triple-Quad mass spectrometer configured with anatmospheric pressure chemical ionisation (APCI) ion source operated inpositive and negative ion mode. The mass spectrometers were scannedbetween m/z 100-1000 with a scan time of 0.3 sec.

¹H NMR spectra were recorded on Varian NMR Spectrometer at 300 MHz, 400MHz or alternatively on a Bruker Avance 500 NMR Spectrometer at 500 MHz.

Unless otherwise specified, HRMS analyses were performed on an Agilent1100 HPLC with an Agilent MSD-TOF mass spectrometer and an Agilent 1100Diode Array Detector using a Zorbax C-18 column (column size: 30×4.6 mm;particle size: 1.8 jm, gradient: 5-95% B in 4.5 min; flow rate: 3.5mL/min; temperature: 70° C., eluents A: 0.05% TFA in H₂O; and eluent B:0.05% TFA in CH₃CN).

Example 1. Preparation of 2,5-difluoro-4-formyl-N-methylbenzamide

Part A. tert-butyl 4-bromo-2,5-difluorobenzoate

A solution of n-butylmagnesium chloride (2 M in THF) (19.9 mL, 39.7mmol) was added to a solution of n-butyllithium (2.5 M in hexanes) (31.8mL, 79.4 mmol) in anhydrous toluene (40 mL) at −10° C. The rate ofaddition was adjusted to keep the internal temperature at less than −5°C. The resulting mixture was stirred at −10° C. for 0.5 hr. Then asolution of 1,4-dibromo-2,5-difluorobenzene (25.4 g, 93.4 mmol) in drytoluene (80 mL) was added at such a rate as to maintain the internaltemperature below −5° C. Afterward, the mixture was stirred at −10° C.for 2 hr. Next, a solution of di-tert-butyl dicarbonate (25.9 g, 0.12mol) in toluene (40 mL) was added at such a rate as to maintain theinternal temperature below −5° C. The mixture was then gradually warmedfrom −10° C. to 10° C. over a period of 2.5 hr. A 10% aqueous solutionof citric acid (175 mL) was then added, and the phases were separated.The organic layer was washed 10% aqueous solution of citric acid (175mL), dried over magnesium sulfate, filtered, and concentrated underreduced pressure to provide tert-butyl 4-bromo-2,5-difluorobenzoate thatwas used directly in the next step.

Part B. 4-bromo-2,5-difluorobenzoic acid

Trifluoromethanesulfonic acid (125 mL) was added to a solution oftert-butyl 4-bromo-2,5-difluorobenzoate (27.4 g, 93.4 mmol) indichloromethane (125 mL) at 0° C. The resulting mixture was stirred atroom temperature 3 hr, and then concentrated under reduced pressure.Half-saturated brine (100 mL) was then added to the residue, and themixture was extracted with dichloromethane (2×90 mL). The organic layerswere combined and the product was extracted with a 1 N aqueous solutionof sodium hydroxide (1×90 mL and 1×50 mL). The combined aqueous layerswere then acidified using a 3 N aqueous solution of hydrochloric acid(80 mL), and the product was extracted with EtOAc (3×100 mL). Thecombined organic layers were over magnesium sulfate, filtered, andconcentrated under reduced pressure to provide4-bromo-2,5-difluorobenzoic acid (13.1 g, 59%) as a solid. ¹H NMR (300MHz, DMSO-d₆) δ ppm 7.78 (dd, J=8.63, 6.23 Hz, 1H), 7.90 (dd, J=9.72,5.58 Hz, 1H), 13.72 (br s, 1H).

Part C. 4-bromo-2,5-difluoro-N-methylbenzamide

Methylamine hydrochloride (3.7 g, 54.9 mmol) and 1-hydroxybenzotriazole(5.99 g, 44.3 mmol) were added to a solution of4-bromo-2,5-difluorobenzoic acid (10 g, 42.2 mmol) inN,N-dimethylformamide (70 mL). At 0° C., triethylamine (8.8 mL, 63.3mmol) and N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride(10.5 g, 54.9 mmol) were then added. The resulting mixture was stirredfrom 0° C. to room temperature for 16 hr. Water (140 mL) was then added,and the product was extracted with EtOAc (3×100 mL). The combinedorganic layers were washed with brine, dried over magnesium sulfate,filtered, and concentrated under reduced pressure. The residue waspurified by silical gel flash chromatography eluting with 10-60% EtOAcin hexanes to give 4-bromo-2,5-difluoro-N-methylbenzamide (9.97 g, 95%)as a solid. ¹H NMR (300 MHz, CDCl₃) δ ppm 3.03 (d, J=4.73 Hz, 3H), 6.70(br s, 1H), 7.38 (dd, J=10.37, 5.23 Hz, 1H), 7.88 (dd, J=8.65, 6.69 Hz,1H).

Part D. 4-Cyano-2,5-difluoro-N-methylbenzamide

Zinc cyanide (2.81 g, 23.9 mmol), zinc dust (0.26 g, 3.99 mmol),tris(dibenzylideneacetone)dipalladium(0) (0.73 g, 0.80 mmol),1,1′-bis(diphenylphosphino)ferrocene (0.88 g, 1.60 mmol) and sodiumacetate (0.13 g, 1.60 mmol) were added to a solution of4-bromo-2,5-difluoro-N-methylbenzamide (9.97 g, 39.9 mmol) inN,N-dimethylformamide (150 mL). N₂ was bubbled into the resultingmixture 5 min and then it was stirred at 100° C. for a period of 2 hr.The resulting mixture was cooled to room temperature and diluted withEtOAc (150 mL). The mixture was filtered on a diatomaceous earth pad,which subsequently was rinsed with EtOAc (2×25 mL). The filtrate waswashed with water (300 mL) and the aqueous layer was back-extracted withEtOAc (2×50 mL) and the combined organic layers were dried with MgSO₄,filtered, and concentrated under reduced pressure. The crude residue waspurified by silical gel flash chromatography eluting with 10-50% EtOAcin hexanes to give 4-cyano-2,5-difluoro-N-methylbenzamide (6.95 g,contains traces of N,N-dimethylformamide) as an solid. ¹H NMR (300 MHz,CDCl₃) δ ppm 3.05 (d, J=4.81 Hz, 3H), 6.78 (br s, 1H), 7.43 (dd,J=10.00, 4.78 Hz, 1H), 7.97 (dd, J=8.79, 5.85 Hz, 1H).

Part E. Preparation of 2,5-difluoro-4-formyl-N-methylbenzamide

Raney nickel (50% in water) (6.38 g) was added to a solution of4-cyano-2,5-difluoro-N-methylbenzamide (6.95 g, 35.4 mmol) in formicacid (94 mL) and water (32 mL). The mixture was stirred at 120° C. for 6hr and then at room temperature for 16 hr. The resulting mixture wasdiluted with methanol (140 mL). Silica gel was added and the slurry wasvigorously stirred 0.25 hr and then filtered on a diatomaceous earthpad. The pad was rinsed with methanol and the filtrate was concentratedunder reduced pressure. The residue was purified by silical gel flashchromatography eluting with 10-60% EtOAc in hexanes to provide2,5-difluoro-4-formyl-N-methylbenzamide (5.25 g, 74%) as a pale yellowsolid. ¹H NMR (300 MHz, CDCl₃) δ ppm 3.06 (d, J=4.84 Hz, 3H), 6.83 (brs, 1H), 7.62 (dd, J=10.84, 5.20 Hz, 1H), 7.97 (dd, J=10.27, 5.62 Hz,1H), 10.34 (d, J=2.98 Hz, 1H); MS (ESI) m/z 200.09 [M+H]⁺.

Example 2. Preparation of 3,5-difluoro-4-formyl-N-methylbenzamide

Part A. Preparation of 3,5-difluoro-4-formylbenzoic acid.

To a solution of 3,5-difluorobenzoic acid (291 g, 1.84 mol) in2-methyltetrahydrofuran (4.35 L) was added TMEDA (604 mL, 4.03 mol) atroom temperature. The resulting solution was cooled to −78° C.Afterward, n-BuLi (2.5 M in hexane) (1.77 L, 4.43 mol) was addeddrop-wise, during which the temperature of the mixture remained at lessthan −65° C. The mixture was then stirred at −78° C. for 1.5 hr.Anhydrous MeOCHO (239 mL, 3.88 mol) was added dropwise at a rate thatallowed the temperature to be maintained at less than −65° C. Theresulting solution was allowed to warm at room temperature, and thenmaintained a room temperature while being stirred for 18 hr. The mixturewas then cooled to 0-5° C., and excess base was quenched with 6M aqueousHCl (2.2 L, 13.2 mol). The phases were then separated, and the aqueouslayer was extracted 3 times with 2-methyltetrahydrofuran (3×500 mL). Thecombined organic phases were washed with saturated brine, dried overMgSO₄, filtered, and concentrated under vacuum. The residue wasdissolved in ethyl acetate (350 mL) at reflux, and cooled to roomtemperature. Hexanes (480 mL) were then added, and the resulting mixturewas further cooled to −15° C. The solid was collected by filtration,rinsed with hexanes, and dried under mechanical vacuum to form the titlecompound (122 g, 35%) as a solid. ¹H NMR (300 MHz, DMSO-d₆) δ ppm7.63-7.70 (m, 2H), 10.23 (s, 1H); MS m/z (ESI) 187.17 [M+H]⁺.

Part B. Preparation of methyl 3,5-difluoro-4-formylbenzoate

K₂CO₃ (14 g, 0.10 mol) and CH₃I (4.6 mL, 74.5 mmol) were added to asolution of 3,5-difluoro-4-formylbenzoic acid (12.6 g, 67.7 mmol) in DMF(135 mL). The resulting mixture was stirred at room temperature for 3hr. Water (200 mL) was then added, and the mixture was extracted withEtOAc (3×150 mL). The combined organic layers were washed with brine,dried with MgSO₄, filtered, and concentrated under reduced pressure. Thecrude residue was purified by silica gel flash chromatography (0 to 20%EtOAc in hexanes) to form methyl 3,5-difluoro-4-formylbenzoate (6 g,44%) as an oil that solidified upon standing. The solid was trituratedin hexanes, filtered, and dried in vacuo to form pure methyl3,5-difluoro-4-formylbenzoate (4.5 g, 33%) as a solid. ¹H NMR (300 MHz,CDCl₃) δ ppm 3.97 (s, 3H), 7.62-7.68 (m, 2H), 10.39 (s, 1H); MS (ESI)m/z 201.30 [M+H]⁺.

Part C. Preparation of 3,5-difluoro-4-formyl-N-methylbenzamide

To an ice-cold solution of 3,5-difluoro-4-formylbenzoic acid (120 g, 645mmol) in dichloromethane (1.5 L) and N,N-dimethylformamide (2.0 g, 27mmol) was added oxalyl chloride (90 g, 709 mmol) drop-wise at a ratethat allowed the mixture to not exceed an internal temperature of 10° C.The resulting mixture was stirred at the same temperature for 0.5 hr,warmed to room temperature, and stirred for an additional 1.5 hr. Thesolution was then cooled to 0° C., and aqueous methylamine (40%, 168 mL,1.94 mol) was added drop-wise at a rate that allowed the mixture to notexceed an internal temperature of 7° C. Afterward, the mixture wasquenched with aqueous HCl (2M, 335 mL, 670 mmol) and warmed to roomtemperature. The organic layer was separated, washed with brine (500mL), dried over MgSO₄, filtered, and concentrated under vacuum. Theresulting residual solid was taken in MTBE (500 mL), and the resultingmixture was heated to reflux for 0.5 hr, cooled to room temperature, andstirred for 18 hr. Afterward, the mixture was cooled to 0° C., filtered,rinsed with pentane, and dried under vacuum to form the title compound(103 g, 80%) as a solid. ¹H NMR (300 MHz, CDCl₃) δ ppm 3.03 (d, J=4.86Hz, 3H), 6.37 (br s, 1H), 7.36-7.42 (m, 2H), 10.36 (s, 1H); MS m/z200.06 [M+H]⁺ (ESI).

Example 3. Preparation of 4-formyl-N,3-dimethylbenzamide

4-Bromo-3-methylbenzoic acid (10 g, 46.5 mmol) was suspended in dry THF(400 mL), purged with N₂, and cooled to 0° C. NaH (60% in oil) (1.95 g,48.8 mmol) was added, and the resulting mixture was stirred at roomtemperature for 20 min. Afterward, the mixture was cooled to −78° C.tert-Butyllithium (1.7 M in pentane, 64 mL, 97.7 mmol) was then added ata rate that allowed the internal temperature to be maintained at lessthan −74° C. After the addition was complete, dry DMF (7.2 mL, 93 mmol)was added. The resulting mixture was stirred at −78° C. for 1 hr andthen allowed to warm up to room temperature. After 1.5 hr, the excessbase was quenched using 1 M HCl aqueous solution until the mixture hadan acidic pH. The mixture was then extracted with EtOAc (3×200 mL), andthe combined organic extracts were dried over MgSO₄, filtered, andconcentrated under reduced pressure to afford 4-formyl-3-methyl benzoicacid. The acid was dissolved in DMF (30 mL), and MeNH₂—HCl (4.08 g, 60.5mmol) and HOBt (6.91 g, 51.2 mmol) were added. The resulting mixture wascooled to 0° C., and then Et₃N (26 mL, 0.19 mol) was added, followed byEDC-HCl (11.6 g, 60.5 mmol). The mixture was stirred at room temperaturefor 16 hr, and then filtered on a silica gel pad, which was subsequentlyrinsed with EtOAc. The filtrate was concentrated under reduced pressure,and the residue was purified by silica gel flash column chromatography(Hex/EtOAc 1:1 to 1:2) to provide the title compound (4.5 g, 55%) as asolid. ¹H NMR (300 MHz, CDCl₃) δ 2.43 (s, 3H), 3.00 (d, J=4.9 Hz, 3H),6.12 (m, 1H), 7.39 (dd, J=8.2, 2.3 Hz, 1H), 7.57 (d, J=8.2 Hz, 1H), 7.64(d, J=2.1 Hz, 1H).

Example 4. Alternative Preparation of 4-formyl-N,3-dimethylbenzamide

Part A. Preparation of 4-cyano-N,3-dimethylbenzamide

A mixture of 4-bromo-N-ethyl-3-methylnenzamide (3.76 g, 16.5 mmol, canbe synthesized according to the following reference Oxford, A. W.; et alEP 533266 (1993)), K₄[Fe(CN)₆]-3H₂O (2.09 g, 4.94 mmol), Na₂CO₃ (1.05 g,9.89 mmol), Pd(OAc)₂ (75 mg, 0.33 mmol), 1,4-diazabicyclo[2.2.2]octane(74 mg, 0.66 mmol), and DMA (20 mL) was maintained under N₂ atmosphereat 126-130° C. for 7.5 hr. Afterward, the mixture was cooled to roomtemperature, diluted with EtOAc, stirred for 20 min, and filteredthrough diatomaceous earth. The filtrate was concentrated under reducepressure, and the residue (5.52 g) was stirred in a mixture of Et₂O (5mL) and hexanes (10 mL). Afterward, the solid was collected byfiltration, and washed with Et₂O to form the title compound (2.53 g).The mother liquor was concentrated under reduce pressure, and theresidue was stirred in a mixture of Et₂O (2 mL) and hexanes (4 mL) toform an additional amount of the compound (0.265 g) as a solid. Bothbatches were combined (2.80 g, 97%). ¹H NMR (300 MHz, CDCl₃) δ ppm 2.59(s, 3H), 3.03 (d, J=4.9 Hz, 3H), 6.17 (br s, 1H), 7.61 (dd, J=8.2, 1.5Hz, 1H), 7.66 (d, J=8.0 Hz, 1H), 7.73 (s, 1H).

Part B. Preparation of 4-formyl-N,3-dimethylbenzamide

To a mixture of Raney Nickel 28000 (wet, 2.02 g) in 75% formic acid (40mL) was added 4-cyano-N,3-dimethylbenzamide (1.94 g, 11.1 mmol). Theresulting mixture was maintained at 100° C. for 3 hr, and then cooled,filtered through diatomaceous earth, and washed with EtOAc and MeOH. Thefiltrate was concentrated under reduced pressure, and the residue waspurified by silica gel flash column chromatography (EtOAc 100%) toafford the title compound (1.94 g, 69%) as a solid.

Example 5. Preparation of N-ethyl-4-formyl-3-methylbenzamide

Ethanamine (0.041 g, 0.91 mmol) was added to 4-formyl-3-methylbenzoicacid (0.15 g, 0.91 mmol) in THF (10 ml) under N₂. The resultingsuspension was stirred for 30 min then4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholin-4-ium (0.243 g,1.01 mmol) was added and the suspension stirred for an additional 18 hr.The resulting mixture was concentrated under reduced pressure and theresidue was dissolved in EtOAc (20 mL) and washed sequentially withwater and brine. The organic layer was dried over MgSO₄, filtered, andconcentrated under reduced pressure. The residue was purified by flashsilica chromatography, eluting with a gradient 0-10% of MeOH in CH₂Cl₂to yield N-ethyl-4-formyl-3-methylbenzamide (0.206 g) which contained˜10-20% of an impurity. The material was used without furtherpurification. LCMS(ES+) m/z calc. for C₁₁H₁₄NO₂ [M+H]⁺ 192.23, found192.27.

Example 6. Preparation of 2-fluoro-4-formyl-N,5-dimethylbenzamide

Part B. Part A. Preparation of 4-bromo-5-fluoro-2-methylbenzaldehyde

A solution of 5-bromo-4-fluoro-2-iodotoluene (5 g, 15.9 mmol) in Et2O(15 mL) was added dropwise to a solution of n-BuLi (2.5 M in hexane)(7.3 mL, 15.9 mmol) in Et2O (10 mL) at −100° C., during which thetemperature of the mixture remained less than −95° C. After 15 min,anhydrous DMF (1.35 mL, 17.5 mmol) was added dropwise. The resultingmixture was warmed from −100° C. to room temperature over 2.5 hr whilestirring. Afterward, 1 N HCl (50 mL) aqueous solution was added toadjust the pH to 1. The resulting mixture was extracted with EtOAc (2×50mL). The combined organic layers were washed with brine, dried overMgSO4, filtered, and concentrated in vacuo. The residue was purified bysilica gel flash chromatography (0-10% EtOAc in hexanes) to obtain4-bromo-5-fluoro-2-methylbenzaldehyde (2.9 g, 84%) as a solid. 1H NMR(300 MHz, CDCl₃): δ ppm 2.63 (s, 3H), 7.50 (d, J=6.5 Hz, 1H), 7.54 (d,J=8.4 Hz, 1H), 10.20 (d, J=1.8 Hz, 1H).

Part B. Preparation of methyl 2-fluoro-4-formyl-5-methyl benzoate

Et₃N (3.5 ml, 25 mmol) was added to a solution of4-bromo-5-fluoro-2-methyl-benzaldehyde (1.08 g, 5.00 mmol) in DMF (20mL) and MeOH (20 mL). CO was bubbled through the resulting solution for10 min, and then PdCl₂(Ph₃P)₂ (0.35 g, 0.50 mmol) was added. Afterward,CO was bubbled through the solution for an additional 10 min, and thenthe mixture was heated at 70° C. for 18 hr under a CO atmosphere. Thesolution was cooled to room temperature, and concentrated under reducedpressure. The residue was diluted with EtOAc (100 mL), washed with asaturated aqueous solution of NaHCO₃ (30 mL), washed with brine (30 mL),dried over Na₂SO₄, filtered, and evaporated under reduced pressure. Theresidue was purified by silica gel flash column chromatography (10-20%EtOAc in hexanes) to form the title compound as a solid (0.403 g, 41%).¹H NMR (400 MHz, CDCl₃) δ ppm 2.67 (s, 3H), 3.97 (s, 3H), 7.57 (d,J=10.4 Hz, 1H), 7.83 (d, J=6.4 Hz, 1H), 10.29 (d, J=1.6 Hz, 1H).

Part C. Preparation of 2-fluoro-4-formyl-N,5-dimethylbenzamide

A mixture of methyl 2-fluoro-4-formyl-5-methylbenzoate (0.35 g, 1.65mmol) in DMF (5 mL) and methanamine 40% wt in water (4.96 mL, 57.64mmol) was stirred at 50° C. for 5 hr. The resulting mixture wasconcentrated under reduced pressure and the residue purified by flashchromatography on silica gel, eluting with a mixture of ethyl acetate(40-100%) and heptane to provide 2-fluoro-4-formyl-N,5-dimethylbenzamide(0.297 g, 92%) as solid. MS m/z (ESI) 195.91 [M+H]⁺.

Example 7. Preparation of (S)-tert-Butyl2-ethynylmorpholine-4-carboxylate

Part A. Preparation of Tosyl Azide

To a solution of sodium azide (37.5 g, 0.577 mol) in water (100 mL) wasadded ethanol (200 mL) at 20° C. To this solution was added a warmsolution (40-45° C.) of tosyl chloride (100.0 g, 0.525 mol) in ethanol(500 mL) over 10 min. The resulting suspension was stirred at 20-25° C.for 2.5 hr. The ethanol was evaporated under reduced pressure and theresidue was taken-up in water (600 mL). The oily product was separatedand the aqueous layer was extracted with dichloromethane (150 mL). Thecombined organic phases were washed with water (2×100 mL), dried oversodium sulfate and evaporated under reduced pressure to give tosyl azide(98.0 g, 95% yield) as an oil. ¹H NMR (400 MHz, CDCl₃) δ ppm 2.49 (s,3H), 7.43 (d, J=8.30 Hz, 2H), 7.85 (d, J=8.30 Hz, 2H). Reference: Org.Synth. Coll. Vol. V, p 179.

Part B. Preparation of dimethyl (1-diazo-2-oxopropyl)phosphonate

To a solution of dimethyl (2-oxopropyl)phosphonate (91.7 g, 0.552 mol)in acetonitrile (920 mL) was added potassium carbonate (91.6 g, 0.662mol). The suspension was stirred at 40° C. for 1 hr. A solution of tosylazide (114.4 g, 0.58 mol) in acetonitrile (460 mL) was then added dropwise over 45 min. The temperature was maintained between 18° C. and 24°C. during the addition. The resulting mixture was stirred for anadditional 2 hr at 20-25° C. and was then filtered over diatomaceousearth. The filer cake was rinsed with acetonitrile (2×100 mL) and thecombined filtrates were evaporated under reduced pressure. The residuewas purified by chromatography (silica, eluting with a mixture of ethylacetate/heptanes) to yield dimethyl (1-diazo-2-oxopropyl)phosphonate(90.5 g, 85%) as an oil. ¹H NMR (400 MHz, CDCl₃) δ ppm 2.26 (s, 3H),3.82 (s, 3H), 3.85 (s, 3H).

Part C. Preparation of tert-butyl (2R)-2-formylmorpholine-4-carboxylate

A solution of tert-butyl (2R)-2-(hydroxymethyl)morpholine-4-carboxylate(91.0 g, 0.418 mol), TEMPO (0.654 g, 0.004 mol), sodium bromide in water(0.5 M, 84 mL, 0.04 mol) and dichloromethane (910 mL) was cooled to 0-5°C. The pH of a solution of sodium hypochlorite (1.66 M, 308 mL, 0.52mol) was adjusted to pH=9.3 the addition of sodium hydrogencarbonate (21g, 0.21 mol), and the resulting solution was added drop wise over 30 minto the resulting mixture. The temperature was kept between 0 and 5° C.during the addition using an ice bath for cooling. The biphasic mixturewas then stirred for an additional 30 min at 0-5° C. The temperature wasadjusted to 20° C. and water (450 mL) was added. The phases wereseparated and the aqueous phase was extracted with dichloromethane(2×180 mL). The combined organic phases were washed with water (2×180mL), then dried over sodium sulfate and evaporated under reducedpressure to give tert-butyl (2R)-2-formylmorpholine-4-carboxylate (63.02g, 70%) as an orange viscous oil. ¹H NMR (400 MHz, CDCl₃) δ ppm 1.47 (s,9H), 2.47-3.13 (m, 2H), 3.37-3.72 (m, 2H), 3.72-4.19 (m, 3H), 9.65 (s,1H).

Part D. Preparation of (S)-tert-butyl 2-ethynylmorpholine-4-carboxylate

To a solution of dimethyl (1-diazo-2-oxopropyl)phosphonate (64.7 g,0.337 mol) in a mixture of acetonitrile (526 mL) and methanol (105 mL)was added potassium carbonate (80.9 g, 0.585 mol). The suspension wasstirred at 18-20° C. for 15 min. A solution of tert-butyl(2R)-2-formylmorpholine-4-carboxylate (63.0 g, 0.293 mol) in a mixtureof acetonitrile (53 mL) and methanol (10 mL) was then added drop wiseover 1 hr while maintaining the temperature between 18 and 23° C. Theresulting mixture was stirred for 2 hr at 20° C. after the end of theaddition and was then held overnight. The resulting suspension wasfiltered and the filtrate was evaporated under reduced pressure. Theresulting oil was added slowly to water (950 mL), and the precipitatewas collected by filtration, and the filter cake was washed with water(120 mL). The residue was purified by chromatography on silica gel(eluting with 10% ethyl acetate in heptanes) to give (S)-tert-butyl2-ethynylmorpholine-4-carboxylate (45.25 g, 64% yield) as a solid. ¹HNMR (400 MHz, CDCl₃) δ ppm 1.47 (s, 9H), 2.49 (s, 1H), 3.20-3.32 (m,2H), 3.49-3.61 (m, 2H), 3.70-3.90 (m, 1H), 3.90-3.99 (m, 1H), 4.21-4.28(m, 1H); chiral HPLC (Chiracel OB-H 0.46×250 mm, hexanes 93%—ethanol 3%,30° C., 0.6 mL/min, isocratic 30 min) 97.0% desired enantiomer.

Example 8. (R)-tert-Butyl 2-ethynylmorpholine-4-carboxylate

This compound was prepared using procedures similar to those describedin Example 6. ¹H NMR (300 MHz, CDCl₃) δ ppm 1.45 (s, 9H), 2.47 (d,J=1.81 Hz, 1H), 3.17-3.37 (m, 2H), 3.45-3.62 (m, 2H), 3.70-3.87 (m, 1H),3.88-3.98 (m, 1H), 4.20-4.26 (m, 1H); MS (ESI) m/z 112.17 [M-Boc+H]⁺.

Example 9. Preparation of (2R,5R)-tert-butyl2-(hydroxymethyl)-5-methylmorpholine-4-carboxylate

Part A. Preparation of (R)-2-(benzylamino)propan-1-ol

Benzaldehyde (10.0 g, 94.2 mmol) and (D)-alaninol (7.08 g, 94.2 mmol)were diluted in dichloroethane (100 mL). The resulting mixture wascooled to 0° C. Acetic acid (5.39 mL, 94.2 mmol) was added, followed 10min later by NaBH(OAc)₃ (9.47 g, 44.7 mmol). After stirring for 18 hr atroom temperature, aqueous Na₂CO₃ was added to adjust the pH to 9, andThis compound was extracted with CH₂Cl₂. The combined organic phaseswere dried over MgSO₄ and concentrated under reduced pressure. Theresidue was purified by flash chromatography on silica gel, eluting withmixtures of 100% CH₂Cl₂ to 10/89/1 MeOH/CH₂Cl₂/NH₄OH to afford the titleproduct (5.30 g, 34%) as an oil. ¹H NMR (300 MHz, CDCl₃) δ ppm 1.08 (d,J=6.3 Hz, 3H), 2.04-2.30 (m, 2H), 2.79-2.89 (m, 1H), 3.28 (dd, J=10.5,6.9 Hz, 1H), 3.59 (dd, J=10.5, 4.2 Hz, 1H), 3.73 (d, J=12.9 Hz, 1H),3.86 (d, J=12.9 Hz, 1H), 7.24-7.36 (m, 5H).

Part B. Preparation of (2R,5R)-4-benzyl-5-methylmorpholin-2-yl)methanol

(R)-2-(Benzylamino)propan-1-ol (5.30 g, 32.1 mmol) was dissolved intoluene (150 mL). (S)-Epichlorohydrin (3.76 mL, 48.1 mmol) was thenadded, followed by LiClO₄ (5.12 g, 48.1 mmol). The resulting mixture wasstirred for 18 hr at room temperature. Sodium methoxide (which wasprepared by adding NaOH (3.85 g, 96.2 mmol) to an ice-cooled solution ofMeOH (60 mL) and stirring for 30 min) was added dropwise to the mixture.After stirring at room temperature for 18 hr, water was added. Toluenewas removed under reduced pressure, and This compound was extracted withCH₂Cl₂. The organic phase was dried over MgSO₄, filtered, andconcentrated under reduced pressure. The residue was purified by flashchromatography on silica gel, eluting with mixtures of Et₂O to 5%MeOH/Et₂O to afford the title compound (5.13 g, 72%) as an oil. ¹H NMR(300 MHz, CDCl₃) δ ppm 1.08 (d, J=6.0 Hz, 3H), 1.90-2.03 (m, 2H),2.36-2.44 (m, 1H), 2.57 (dd, J=11.7, 2.1 Hz, 1H), 3.05 (d, J=13.2 Hz,1H), 3.31-3.64 (m, 4H), 3.77 (dd, J=11.7, 3.3 Hz, 1H), 4.14 (d, J=13.2Hz, 1H), 7.23-7.34 (m, 5H).

Part C. Preparation of (2R,5R)-tert-butyl2-(hydroxymethyl)-5-methylmorpholine-4-carboxylate

(2R,5R)-4-Benzyl-5-methylmorpholin-2-yl)methanol (5.10 g, 23.0 mmol) wasdissolved in MeOH, and N₂ was bubbled through the resulting solution.Boc₂O (5.03 g, 23.0 mmol) was then added, followed by Pd(OH)₂ (2.55 g).After stirring for 18 hr under 1.01 bar H₂, the mixture was diluted withCH₂Cl₂ and filtered through a pad of diatomaceous earth. The filtratewas concentrated under reduced pressure, and the residue was purified byflash chromatography on silica gel, eluting with mixtures of 70% to 80%of EtOAc and hexanes to produce the title compound (3.20 g, 60%) as anoil. ¹H NMR (300 MHz, DMSO-d₆) δ ppm 1.12 (d, J=6.3 Hz, 3H), 1.39 (s,9H), 3.19-3.58 (m, 5H), 3.60-3.74 (m, 2H), 3.81-3.90 (m, 1H), 4.69 (dd,J=6.0, 4.5 Hz, 1H). MS (ESI) m/z 132.3 [M+H-Boc]⁺.

Example 10. Preparation of (2R,5S)-tert-butyl2-(hydroxymethyl)-5-methylmorpholine-4-carboxylate

This compound was prepared using procedures similar to those describedin Example 8. ¹H NMR (300 MHz, CDCl₃) δ ppm 1.22 (d, J=6.86 Hz, 3H),1.46 (s, 9H), 1.89-1.95 (m, 1H), 2.83-3.00 (m, 1H), 3.38-4.04 (m, 7H).

Example 11. tert-butyl 3-ethynylpyrrolidine-1-carboxylate

This compound was synthesized using procedures similar to thosedescribed in Example 6, part D and starting from commercially availablestarting material. ¹H NMR (300 MHz, CDCl₃) δ ppm 1.45 (s, 9H), 1.85-2.00(m, 1H), 2.08-2.20 (m, 2H), 2.87-3.00 (m, 1H), 3.21-3.38 (m, 2H),3.40-3.69 (m, 2H); MS (ESI) m/z 96.15 [M-Boc+H]⁺.

Example 12. tert-butyl 4-ethynyl-2-methylpyrrolidine-1-carboxylate

Part A. Preparation of 1-benzyl-5-methylpyrrolidin-2-one

Sodium hydride (60% suspension in mineral oil) (2.54 g, 63.6 mmol) wasadded to a solution of 5-methylpyrrolidin-2-one (4.2 g, 42.4 mmol) inN,N-dimethylformamide (27 mL) at 0° C. Benzyl bromide (6.05 mL, 50.8mmol) was then added and the resulting mixture was stirred for 16 h,while letting the temperature rise from 0° C. to room temperature. Asaturated aqueous solution of ammonium chloride (50 mL) was then slowlyadded and the product was extracted with ethyl acetate (3×50 mL). Thecombined organic layers were washed with a 5% aqueous solution of sodiumhydrogen carbonate (100 mL), dried with magnesium sulfate, filtered andconcentrated under reduced pressure. The residue was purified bychromatography on a 100 g silica gel cartridge using a gradient of 5 to75% of EtOAc in hexanes to provide 1-benzyl-5-methylpyrrolidin-2-one(6.88 g, 86%) as an oil. ¹H NMR (300 MHz, CDCl₃): δ ppm 1.16 (d, J=6.25Hz, 3H), 1.53-1.64 (m, 1H), 2.04-2.21 (m, 1H), 2.34-2.56 (m, 2H),3.46-3.58 (m, 1H), 3.98 (d, J=15.02 Hz, 1H), 4.97 (d, J=15.02 Hz, 1H),7.21-7.35 (m, 5H).

Part B. Preparation of methyl1-benzyl-5-methyl-2-oxopyrrolidine-3-carboxylate

n-Butyllithium (2.5 M in hexanes) (29 mL, 72.7 mmol) was slowly added toa solution of diisopropylamine (10.7 mL, 76.3 mmol) in tetrahydrofuran(50 mL) cooled using an ice bath so to keep the internal temperaturebelow 10° C. The resulting mixture was cooled to −78° C. and a solutionof 1-benzyl-5-methylpyrrolidin-2-one (6.88 g, 36.4 mmol) intetrahydrofuran (46 mL) was added at such a rate as to maintain theinternal temperature below −65° C. After the end of the addition, themixture was stirred at −78° C. 0.5 hr. Dimethyl carbonate (6.13 mL, 72.7mmol) was then added, and the mixture was stirred at −78° C. 0.25 hr.The cold bath was then removed, and the mixture was gradually warmed toroom temperature and stirred at this temperature 16 hr. A 1 N aqueoussolution of hydrochloric acid (100 mL) was added and the product wasextracted with ethyl acetate (3×100 mL). The combined organic layerswere washed with water (150 mL) and then brine (150 mL). The organiclayer was dried with magnesium sulfate, filtered and concentrated underreduced pressure. The crude residue was purified by silica gel flashusing a gradient of 10 to 60% of EtOAc in hexanes to furnish methyl1-benzyl-5-methyl-2-oxopyrrolidine-3-carboxylate (6.85 g, 76%) as an oiland as a mixture of diastereoisomers. ¹H NMR (300 MHz, CDCl₃): δ ppm1.16 and 1.25 (2d, J=6.37 and 6.25 Hz, 3H), 1.79-1.89 (m, 0.5H),1.97-2.08 (m, 0.5H), 2.34-2.54 (m, 1H), 3.45-3.74 (m, 2H), 3.79 and 3.81(2 s, 3H), 4.01 (dd, J=14.99, 9.43 Hz, 1H), 4.99 (dd, J=15.01, 11.63 Hz,1H), 7.21-7.36 (m, 5H).

Part C. Preparation of (1-benzyl-5-methypyrrolidin-3-yl)methanol

Lithium aluminum hydride (3.89 g, 0.10 mol) was carefully added toanhydrous tetrahydrofuran (100 mL) at 0° C. A solution of methyl1-benzyl-5-methyl-2-oxopyrrolidine-3-carboxylate (6.85 g, 27.7 mmol) inanhydrous tetrahydrofuran (39 mL) was then added. The resulting mixturewas stirred at room temperature 16 hr, then was cooled to 0° C., andwater (4 mL) was very slowly added, followed by a 15% aqueous solutionof sodium hydroxide (4 mL) and again water (12 mL). The resultingsuspension was stirred at 0° C. 1 hr and then magnesium sulfate wasadded. The suspension was stirred at 0° C. 0.25 hr and filtered on a padof diatomaceous earth. The filter cake was washed with ethyl acetate andthe filtrate was concentrated under reduced pressure to provide(1-benzyl-5-methylpyrrolidin-3-yl)methanol (5.53 g, 97%) as an oil. Theresulting product used in the next step without further purification.

Part D. Preparation of (5-methylpyrrolidin-3-yl)methanol

A solution of (1-benzyl-5-methylpyrrolidin-3-yl)methanol (5.53 g, 26.9mmol) in ethanol (192 mL) was put under vacuum and backfilled withnitrogen three times. Palladium hydroxide on carbon (20 wt. %, 50% wet)(1.89 g) was added. Hydrogen was bubbled into the suspension 0.25 hr.The resulting mixture was stirred under 1.01 bar of hydrogen 16 hr atroom temperature. The mixture was then filtered on a pad of diatomaceousearth. The filter cake was washed with ethanol and the filtrate wasconcentrated under reduced pressure to provide(5-methylpyrrolidin-3-yl)methanol (3.16 g, quant., mixture ofdiastereoisomers) as an oil. ¹H NMR (300 MHz, CDCl₃): δ ppm 0.96-1.06(m, 0.5H), 1.14 and 1.18 (2d, J=6.35 and 6.19 Hz, 3H), 1.37-1.47 (m,0.5H), 1.65-1.74 (m, 0.5H), 2.00-2.15 (m, 3H), 2.27-2.43 (m, 1H), 2.63(dd, J=10.90, 6.50 Hz, 0.5H), 2.86-3.00 (m, 1H), 3.06-3.24 (m, 1H),3.48-3.66 (m, 2H).

Part E. tert-butyl 4-(hydroxymethyl)-2-methylpyrrolidine-1-carboxylate

Potassium carbonate (19 g, 0.14 mol) and di-tert-butyl dicarbonate (5.99g, 27.4 mmol) were added to a solution of(5-methylpyrrolidin-3-yl)methanol (3.16 g, 27.4 mmol) in tetrahydrofuran(30 mL) and water (30 mL) at 0° C. The resulting mixture was stirred atroom temperature 16 hr and then water (50 mL) was added. The product wasextracted with ethyl acetate (3×50 mL). The combined organic layers werewashed with brine (100 mL), dried over magnesium sulfate, filtered andconcentrated under reduced pressure. The residue was purified bychromatography on a 100 g silica gel cartridge using a gradient of 10 to70% of EtOAc in hexanes to furnish tert-butyl4-(hydroxymethyl)-2-methylpyrrolidine-1-carboxylate (4.38 g, 74%,mixture of diastereoisomers). ¹H NMR (300 MHz, CDCl₃): δ ppm 1.14-1.28(m, 3H), 1.30-1.70 (m, 2H), 1.45 (s, 9H), 1.71-1.88 (m, 0.5H), 2.17-2.35(m, 1H), 2.42-2.57 (m, 0.5H), 3.00 (dd, J=10.97, 8.94 Hz, 0.5H),3.05-3.20 (m, 0.5H), 3.50 (dd, J=10.98, 7.64 Hz, 0.5H), 3.55-4.05 (m,3.5H).

Part F. tert-butyl 4-formyl-2-methylpyrrolidine-1-carboxylate

Dess-Martin periodinane (4.23 g, 10.2 mmol) was added to a suspension oftert-butyl 4-(hydroxymethyl)-2-methylpyrrolidine-1-carboxylate (2 g,9.29 mmol) and sodium hydrogencarbonate (1.17 g, 13.9 mmol) indichloromethane (23 mL) at 0° C. The resulting mixture was stirred atroom temperature 2 hr and then a 10% aqueous solution of sodiumthiosulfate (50 mL) and a 5% aqueous solution of sodiumhydrogencarbonate (25 mL) were added. The resulting mixture was stirredat room temperature 16 hr. The phases were separated and the aqueouslayer was extracted with dichloromethane (1×25 mL). The combined organiclayers were dried with magnesium sulfate, filtered and concentratedunder reduced pressure to provide tert-butyl4-formyl-2-methylpyrrolidine-1-carboxylate (2.09 g, quant., mixture ofdiastereoisomers) as an oil. ¹H NMR (300 MHz, CDCl₃): δ ppm 1.13-1.22(m, 3H), 1.46 (s, 9H), 1.71-1.81 (m, 0.5H), 1.86-1.96 (m, 0.5H),2.15-2.40 (m, 1H), 2.88-2.98 (m, 0.5H), 3.00-3.13 (m, 0.5H), 3.48-3.58(m, 0.5H), 3.60-3.75 (m, 1.5H), 3.85-4.05 (m, 1H) 9.65 and 9.73 (2d,J=2.14 and 1.51 Hz, 1H).

Part H. tert-butyl 4-ethynyl-2-methylpyrrolidine-1-carboxylate

Potassium carbonate (2.6 g, 18.8 mmol) was added to a solution oftert-butyl 4-formyl-2-methylpyrrolidine-1-carboxylate (2.0 g, 9.38 mol)in acetonitrile (25 mL) and methanol (5 mL). Dimethyl(1-diazo-2-oxopropyl)phosphonate (2.16 g, 11.3 mmol) was then addeddropwise. The suspension was stirred at room temperature for 16 hr andthen was concentrated under reduced pressure. A 5% aqueous solution ofsodium hydrogencarbonate (25 mL) was added to the crude residue. Theproduct was extracted with ethyl acetate (3×25 mL). The combined organicphases were dried with magnesium sulfate, filtered and concentratedunder reduced pressure. The crude material was purified by silica gelchromatography using a gradient of 0 to 30% of EtOAc in heptanes toyield tert-butyl 4-ethynyl-2-methylpyrrolidine-1-carboxylate (1.31 g,67%, mixture of diastereoisomers) as an oil. ¹H NMR (300 MHz, CDCl₃): δppm 1.16 (d, J=6.21 Hz, 1.5H), 1.31 (br s, 1.5H), 1.46 (s, 9H),1.60-1.72 (m, 0.5H), 1.85 (ddd, J=12.25, 6.41, 2.53 Hz, 0.5H), 2.03-2.18(m, 0.5H), 2.10 (dd, J=5.13, 2.38 Hz, 1H), 2.36-2.45 (m, 0.5H),2.76-2.88 (m, 0.5H), 2.96-3.08 (m, 0.5H), 3.24 (dd, J=10.85, 8.70 Hz,0.5H), 3.28-3.40 (m, 0.5H), 3.59-3.67 (m, 0.5H), 3.74-4.06 (m, 1.5H); MS(ESI) m/z calcd for C₇H₁₂N, 110.10 [M-Boc+H]⁺, found 110.11.

Example 13. (2S,5R)-tert-butyl2-ethynyl-5-methylmorpholine-4-carboxylate

This compound was prepared using procedures similar to those describedin Example 6, part D. ¹H NMR (300 MHz, CDCl₃) δ ppm 1.24 (d, J=6.64 Hz,3H), 1.46 (s, 9H), 2.50 (s, 1H), 3.09 (t, J=11.82 Hz, 1H), 3.66 (q,J=12.23 Hz, 2H), 3.85-3.99 (m, 1H), 4.08 (d, J=7.82 Hz, 2H); MS (ESI)m/z 126.19 [M-Boc+H]⁺.

Example 14. (2S,5S)-tert-butyl2-ethynyl-5-methylmorpholine-4-carboxylate

This compound was prepared using procedures similar to those describedin Example 6, part D. ¹H NMR (300 MHz, CDCl₃) δ ppm 1.24 (d, J=7.01 Hz,3H), 1.47 (s, 9H), 2.42 (s, 1H), 3.29-3.46 (m, 2H), 3.88 (d, J=13.21 Hz,1H), 4.11-4.23 (m, 2H), 4.55 (s, 1H); MS (ESI) m/z 126.21 [M-Boc+H]⁺.

Example 15. Preparation of(S)-methyl-2-((2-(2,6-difluoro-4-(methylcarbamoyl)phenyl)-7-methylimidazo[1,2-a]pyridin-3-yl)methyl)morpholine-4-carboxylate

Part A. Preparation of (S)-tert-butyl2-((2-(2,6-difluoro-4-(methylcarbamoyl)phenyl)-7-methylimidazo[1,2-a]pyridin-3-yl)methyl)morpholine-4-carboxylate.Also, illustrates General Cyclization Conditions 1

A mixture of 4-methylpyridin-2-amine (5.41 g, 0.050 mol),3,5-difluoro-4-formyl-N-methylbenzamide (9.96 g, 0.050 mol),(S)-tert-butyl 2-ethynylmorpholine-4-carboxylate (10.57 g, 0.050 mol),copper(I) chloride (1.49 g, 0.015 mol),bis(trifluoromethylsulfonyloxy)copper (5.42 g, 0.015 mol) and toluene(120 mL) was charged to a 250 mL jacketed reactor under N₂. Heating wasapplied and the temperature reached 85° C. in 5 min.N,N-dimethylacetamide (1.0 mL) was then added and the resulting mixturewas stirred at 85° C. for 5 hr. The resulting mixture was then cooled to20° C. and held overnight at this temperature. The toluene layer wasseparated by decantation from a solid residue, and then was concentratedunder reduced pressure. The resulting residue was mixed with the solidresidue isolated from decantation, and the combined solids weredissolved dichloromethane (300 mL). A solution of sodium sulfide inwater (18% w/w) was added to the organic solution and the mixture wasstirred for 15 min. The mixture was then filtered over a pad ofdiatomaceous earth and the cake was washed with dichloromethane (2×160mL). The layers were separated and the aqueous phase was extracted withdichloromethane (100 mL). The combined organic layers were concentratedunder reduced pressure. The residue was dissolved with ethyl acetate(500 mL), combined with silica gel (50 g), stirred for 30 min and thenfiltered over a pad of diatomaceous earth. The solution was concentratedunder reduced pressure and the residue dissolved with dichloromethane(300 mL). Silica gel (32.5 g) was added and the mixture was concentratedto dryness under reduced pressure. The pre-absorbed crude material waspacked on a dry silica column (80 g) and was eluted with ethylacetate-heptane 50-50 v/v to give 13.7 g of a residue material which wasthen triturated in a mixture of ethyl acetate-heptane 30-70 v/v. Thesolid was collected by filtration, the filter cake washed with ethylacetate-heptane 30-70 v/v, and the resulting solid dried mechanicalvacuum to yield (S)-tert-butyl2-((2-(2,6-difluoro-4-(methylcarbamoyl)phenyl)-7-methylimidazo[1,2-a]pyridin-3-yl)methyl)morpholine-4-carboxylateas a white solid (11.0 g, 44%). ¹H NMR (400 MHz, DMSO-d₆) δ ppm 1.35 (s,9H), 2.36-2.81 (m, 5H), 2.85 (d, J=4.46 Hz, 3H), 3.06 (d, J=6.12 Hz,2H), 3.24 (t, J=10.93 Hz, 1H), 3.43-3.50 (m, 1H), 3.56-3.72 (m, 3H),6.85 (d, J=6.84 Hz, 1H), 7.37 (s, 1H), 7.68 (d, J=7.89 Hz, 2H), 8.44 (d,J=6.84 Hz, 1H), 8.72 (br. s, 1H); MS (ESI) m/z 501.16 [M+H]⁺. Forfurther discussion related to this method, see Chernyak, N, et al.,“General and Efficient Copper-Catalyzed Three-Component CouplingReaction towards Imidazoheterocycles. One-Pot Synthesis of Alpidem andZolpidem,” Angewandte Chemie, vol. 49(15), pp. 2743-2746 (Int'l Ed.2010).

Part B. Preparation of(S)-methyl-2-((2-(2,6-difluoro-4-(methylcarbamoyl)phenyl)-7-methylimidazo[1,2-a]pyridin-3-yl)methyl)morpholine-4-carboxylate

To a solution(S)-tert-butyl-2-((2-(2,6-difluoro-4-(methylcarbamoyl)phenyl)-7-methylimidazo[1,2-a]pyridin-3-yl)methyl)morpholine-4-carboxylate(41.0 g, 0.082 mol) in methanol (330 mL) was added a solution of 3 Nhydrogen chloride in methanol (273 mL, 0.819 mol) over 15 min at 20-25°C. The resulting mixture was heated to 40-45° C. and stirred for 1 hr.The resulting mixture was then cooled to 20° C. and concentrated underreduced pressure. The residue was dissolved with dichloromethane (410mL), and N,N-diisopropylethylamine (26.5 g, 0.205 mol) was added. Methylchloroformate (9.3 g, 0.098 mol) was then added drop wise at 20° C.followed by additional N,N-diisopropylethylamine (5.3 g, 0.040 mol).After stirring for 16 h, the organic layer was washed with water (100mL). The aqueous phase was extracted with dichloromethane (50 mL), thecombined organic phases were then washed with aqueous potassiumhydrogencarbonate 20% w/w and concentrated under reduced pressure. Theresidue was recrystallized from ethyl acetate (860 mL) with a hotfiltration step to remove insoluble material. The precipitate wascollected by filtration and the cake was rinsed with ethyl acetate (2×40mL) and then dried under mechanical vacuum at 50° C. to yield the titlecompound as an ethyl acetate solvate (white solid, 31.8 g). Ethylacetate was removed as follows: the material was dissolved in water (320mL) by gradual addition of hydrochloric acid 3 N (22.5 mL) and theresulting solution was filtered over a filter paper to remove insolublematerial. Ethyl acetate was removed by evaporation under reducedpressure until about 75 mL of mixed ethyl acetate-water had beencollected in the receiver. Water (75 mL) was added to replace the volumedistilled. The solution was transferred to a round bottom flask equippedwith mechanical stirring and the pH was gradually adjusted to 8.5 by aslow addition of aqueous potassium hydrogencarbonate 20% w/w (135 mL).The resulting suspension was stirred overnight at 20-25° C. The productwas collected by filtration and the cake was washed with water (30 mL)and then dried in a vacuum oven to yield(S)-methyl-2-((2-(2,6-difluoro-4-(methylcarbamoyl)phenyl)-7-methylimidazo[1,2-a]pyridin-3-yl)methyl)morpholine-4-carboxylate(25.25 g, 67%) as a solid. ¹H NMR (400 MHz, DMSO-d₆) δ ppm 2.39 (s, 3H),2.85 (s, 4H), 3.07 (br s, 2H), 3.25 (m, 1H), 3.45-3.79 (m, 8H), 6.85 (brs, 1H), 7.37 (br s, 1H), 7.68 (br s, 2H), 8.45 (br s, 1H), 8.72 (br s,1H); HRMS m/z calcd for C₂₃H₂₅F₂N₄O₄ 459.1838 [M+H]⁺, found 459.1844.

Example 15B. Preparation of (R)-tert-butyl2-((7-methyl-2-(2-methyl-4-(methylcarbamoyl)phenyl)imidazo[1,2-a]pyridin-3-yl)methyl)morpholine-4-carboxylate.Illustrates General Cyclization Conditions 2

A 0.5-2.0 mL microwave vial was charged with 4-methylpyridin-2-amine (1eq.), 4-formyl-N,3-dimethylbenzamide (1.05 eq.), (R)-tert-butyl2-ethynylmorpholine-4-carboxylate (1 eq.), copper(I) chloride (0.05eq.), bis(trifluoromethylsulfonyloxy)copper (0.05) and toluene (4 mL).The resulting mixture was purged for 5 min with N₂. The resultingmixture was stirred at 140° C. under microwave irradiation for 45 min.The resulting mixture was dissolved in EtOAc and was filtered usingfluted filter paper. The filtrate was concentrated under reducedpressure and the residue was purified by silica gel flash chromatographyeluting with 2% to 20% ethyl acetate in methanol to provide(R)-tert-butyl2-((7-methyl-2-(2-methyl-4-(methylcarbamoyl)phenyl)imidazo[1,2-a]pyridin-3-yl)methyl)morpholine-4-carboxylate(30.3%) as an oil. ¹H NMR (400 MHz, CD₃OD) δ ppm 1.32-1.48 (m, 10H),2.24 (s, 3H), 2.39 (s, 3H), 2.46-2.59 (m, 1H), 2.70-2.91 (m, 1H), 2.95(s, 3H), 2.98-3.10 (m, 2H), 3.43-3.58 (m, 1H), 3.60-3.79 (m, 3H), 6.88(d, J=7.03 Hz, 1H), 7.04 (br. s., 1H), 7.40 (d, J=7.42 Hz, 1H), 7.70 (d,J=7.42 Hz, 1H), 7.79 (s, 1H), 8.37 (d, J=5.47 Hz, 1H). MS(ESI) m/z479.18 [M+H]⁺.

Example 15C. Preparation of (S)-tert-butyl2-((7-methyl-2-(2-methyl-4-(methylcarbamoyl)phenyl)imidazo[1,2-a]pyridin-3-yl)methyl)morpholine-4-carboxylate.Also illustrates General Cyclization Conditions 3

A mixture of 4-methylpyridin-2-amine (1 eq.), copper(I) chloride (0.05eq.), bis(trifluoromethylsulfonyloxy)copper (0.05 eq.) and4-formyl-N,3-dimethylbenzamide (1 eq.) under N₂ was stirred at roomtemperature for 5 min. Degassed toluene (2 mL), (S)-tert-butyl2-ethynylmorpholine-4-carboxylate (1.5 eq.) were added, and theresulting mixture was stirred at 120° C. for 17 hr. The resultingmixture was cooled to rt, concentrated under reduced pressure and theresidue was purified by silica gel flash chromatography eluting with 1%to 10% methanol in ethyl acetate to provide (S)-tert-butyl2-((7-methyl-2-(2-methyl-4-(methylcarbamoyl)phenyl)imidazo[1,2-a]pyridin-3-yl)methyl)morpholine-4-carboxylate(174 mg, 56.2%) as an oil. MS (ESI) m/z 479.58 [M+H]⁺.

Example 15D. Preparation of (R)-tert-butyl2-((2-(2,6-difluoro-4-(methylcarbamoyl)phenyl)-7-methylimidazo[1,2-a]pyridin-3-yl)methyl)morpholine-4-carboxylate.Also illustrates General Cyclization Conditions 4

A mixture of 4-methylpyridin-2-amine (1 eq.),3,5-difluoro-4-formyl-N-methylbenzamide (1 eq.), (R)-tert-butyl2-ethynylmorpholine-4-carboxylate (1 eq.), copper(I) chloride (0.03 eq.)and bis(trifluoromethylsulfonyloxy)copper (0.03 eq.) under N₂ in toluene(4 mL) and DMA (0.1 mL) was stirred at room temperature for 5 min. Theresulting mixture was stirred at 85° C. for 18 hr. The resulting mixturewas cooled to rt, concentrated under reduced pressure and the residuewas purified by silica gel flash chromatography eluting with 2% to 10%methanol in ethyl acetate to provide (R)-tert-butyl2-((2-(2,6-difluoro-4-(methylcarbamoyl)phenyl)-7-methylimidazo[1,2-a]pyridin-3-yl)methyl)morpholine-4-carboxylate(65.5%) as an oil. MS (ESI) m/z 501.77 [M+H]⁺.

Example 15E. Preparation of (2S,5S)-tert-Butyl5-methyl-2-((7-methyl-2-(2-methyl-4-(methylcarbamoyl)phenyl)imidazo[1,2-a]pyridin-3-yl)methyl)morpholine-4-carboxylate.Also illustrates General Cyclization Conditions 5

4-Methylpyridin-2-amine (0.14 g, 1.26 mmol),4-formyl-N,3-dimethylbenzamide (0.22 g, 1.26 mmol), (2S,5S)-tert-butyl2-ethynyl-5-methylmorpholine-4-carboxylate (0.28 g, 1.26 mmol), andcopper(I) chloride (62 mg, 0.63 mmol) were added to a round-bottomflask. Toluene (10 mL) and N,N-dimethylacetamide (5 drops) were thenadded. The flask was put under vacuum and then back-filled with N₂ threetimes. The resulting mixture was stirred at 90° C. for 16 hr, and thencooled to room temperature. Afterward, ethanol and silica gel were addedto the mixture, which was then concentrated under reduced pressure. Theresidue was purified by flash-chromatography on a 50 g silica gelcartridge eluting using 50% to 100% ethyl acetate in hexanes as eluent,followed by 0% to 20% methanol in ethyl acetate, to provide(2S,5S)-tert-butyl5-methyl-2-((7-methyl-2-(2-methyl-4-(methylcarbamoyl)phenyl)imidazo[1,2-a]pyridin-3-yl)methyl)morpholine-4-carboxylate(0.37 g, 60%) as an oil which was used without further purification.

The compounds of Examples 16-44 in the following Table 1 were preparedusing processes similar to those described in Examples 15, 15B, 15C,15D, and 15E using corresponding starting materials and one of thegeneral cyclization conditions as indicated below.

TABLE 1 EXAMPLES 18-46 Cycli- zation Pro- Ex Compound Structure cedureLCMC (RT, m/z)¹ ¹H NMR 16

1 HRMS (ESI) m/z calcd for C₂₄H₂₆F₂N₄O₃ 457.2046 [M + H]⁺, found457.2042. ¹H NMR (400 MHz, CD3OD) δ ppm 1.04 (dt, J = 9.77, 7.42 Hz, 3H), 2.20- 2.43 (m, 2 H), 2.45 (s, 3 H), 2.60-2.90 (m, 1 H), 2.95 (s, 3H), 3.05-3.18 (m, 3 H), 3.25-3.45 (m, 2H), 3.47-3.64 (m, 1 H), 3.70 (t,J = 12.11 Hz, 1 H), 3.75-3.85 (m, 1 H), 4.18-4.31 (m, 1 H), 6.87 (td, J= 4.69, 2.34 Hz, 1 H), 7.33 (s, 1 H), 7.59 (dd, J = 8.20, 5.86 Hz, 2 H),8.36-8.46 (m, 1 H). 17

2 HRMS m/z calcd for C₂₅H₃₀FN₄O₃ 453.2301 [M + H]⁺, found 453.2296. ¹HNMR (400 MHz, DMSO-d₆) δ ppm 0.88-0.99 (m, 3 H), 2.12-2.35 (m, 5 H),2.38 (s, 3 H), 2.53-2.64 (m, 1 H), 2.79 (d, J = 4.30 Hz, 3 H), 2.92-3.13(m, 2 H), 3.14-3.30 (m, 2 H), 3.40- 3.65 (m, 2 H), 3.67-3.84 (m, 1 H),4.02-4.21 (m, 1 H), 6.77-6.88 (m, 1 H), 7.23-7.40 (m, 2 H), 7.54 (d, J =7.42 Hz, 1 H), 8.27 (br. s., 1 H), 8.43 (dd, J = 17.38, 7.23 Hz, 1 H).18

2 HRMS m/z calcd for C₂₄H₂₈FN₄O₃ 439.214 [M + H]⁺, found 439.2143. ¹HNMR (400 MHz, CD₃OD) δ ppm 2.05 (2 s, 3 H), 2.25 (s, 3 H), 2.41 (dd, J =12.89, 10.94 Hz, 1 H), 2.46 (s, 3 H), 2.70 (d, J = 3.52 Hz, 1 H), 3.03-3.10 (m, 3 H), 3.12-3.24 (m, 2 H), 3.34-3.45 (m, 1 H), 3.48-3.61 (m, 2H), 3.62-3.86 (m, 1 H), 4.13-4.32 (m, 1 H), 6.87 (dt, J = 7.13, 1.90 Hz,1 H), 7.16-7.29 (m, 1 H), 7.29-7.36 (m, 1 H), 7.70 (dd, J = 7.62, 3.71Hz, 1 H), 8.37 (dd, J = 15.23, 7.03 Hz, 1 H). 19

2 HRMS m/z calcd for C₂₄H₂₈FN₄O₃ 439.214 [M + H]⁺, found 439.2142. ¹HNMR (400 MHz, CD₃OD) δ ppm 2.05 (2 s, 3 H), 2.25 (s, 3 H), 2.41 (dd, J =12.89, 10.94 Hz, 1 H), 2.46 (s, 3 H), 2.70 (d, J = 3.52 Hz, 1 H), 3.03-3.10 (m, 3 H), 3.12-3.24 (m, 2 H), 3.34-3.45 (m, 1 H), 3.48-3.61 (m, 2H), 3.62-3.86 (m, 1 H), 4.13-4.32 (m, 1 H), 6.87 (dt, J = 7.13, 1.90 Hz,1 H), 7.16-7.29 (m, 1 H), 7.29-7.36 (m, 1 H), 7.70 (dd, J = 7.62, 3.71Hz, 1 H), 8.37 (dd, J = 15.23, 7.03 Hz, 1 H). 20

3 HRM m/z calcd for C₂₄H₂₉N₄O₃ 421.2234 [M + H]⁺, found 421.224. ¹H NMR(400 MHz, METHANOL-d₄) δ ppm 2.02 (2 s, 3 H), 2.30 (s, 3 H), 2.38 (dd, J= 13.09, 10.74 Hz, 1 H), 2.45 (s, 3 H), 2.60-2.74 (m, 1 H), 2.84- 2.95(m, 3 H), 3.03-3.10 (m, 2 H), 3.10-3.22 (m, 1 H), 3.59-3.70 (m, 2 H),3.81 (dd, J = 11.91, 3.32 Hz, 1 H), 4.21 (d, J = 13.28 Hz, 1 H), 6.86(d, J = 7.03 Hz, 1 H), 7.31 (s, 1 H), 7.40- 7.51 (m, 1 H), 7.66-7.76 (m,1 H), 7.79 (br. s., 1 H), 8.37 (dd, J = 12.11, 7.03 Hz, 1 H). 21

3 HRMS m/z calcd for C₂₄H₂₉N₄O₄ 437.2183 [M + H]⁺, found 437.2177. ¹NMR(400 MHz, DMSO-d₆) δ ppm 2.26 (s, 3 H), 2.37 (s, 3 H), 2.41-2.48 (m, 1H), 2.80 (d, J = 4.30 Hz, 4 H), 3.02 (d, J = 6.64 Hz, 2 H), 3.25 (td, J= 11.72, 2.73 Hz, 1 H), 3.32 (s, 3 H), 3.44-3.53 (m, 1 H), 3.58-3.76 (m,3 H), 6.80 (dd, J = 7.23, 1.76 Hz, 1 H), 7.33 (s, 1 H), 7.40 (d, J =8.20 Hz, 1 H), 7.69 (dd, J = 7.81, 1.56 Hz, 1 H), 7.77 (s, 1 H), 8.39(d, J = 7.03 Hz, 1 H), 8.46 (d, J = 4.69 Hz, 1 H). 22

3 HRMS m/z calcd for C₂₄H₂₉N₄O₄ 437.2183 [M + H]⁺, found 437.2177. ¹HNMR (400 MHz, DMSO-d₆) δ ppm 2.26 (s, 3 H), 2.37 (s, 3 H), 2.80 (d, J =4.30 Hz, 4 H), 3.02 (d, J = 6.64 Hz, 2 H), 3.17-3.30 (m, 1 H), 3.42-3.58(m, 5 H), 3.59-3.75 (m, 3 H), 6.80 (dd, J = 7.23, 1.76 Hz, 1 H), 7.33(s, 1 H), 7.40 (d, J = 7.81 Hz, 1 H), 7.69 (dd, J = 7.81, 1.56 Hz, 1 H),7.77 (s, 1 H), 8.39 (d, J = 7.03 Hz, 1 H), 8.46 (d, J = 4.30 Hz, 1 H) 23

3 HRM m/z calcd for C₂₃H₂₅F₂N₄O₃ 443.1889 [M + H]⁺, found 443.1897. ¹HNMR (400 MHz, CD₃OD) δ ppm 2.06 (d, J = 1.56 Hz, 3 H), 2.39-2.77 (m, 4H), 2.90-3.05 (m, 3 H), 3.10- 3.25 (m, 3 H), 3.36-3.46 (m, 1 H),3.54-3.89 (m, 3 H), 4.20-4.37 (m, 1 H), 6.86 (d, J = 7.03 Hz, 1 H), 7.34(br. s., 1 H), 7.49-7.59 (m, 1 H), 7.59- 7.67 (m, 1 H), 8.42 (dd, J =14.06, 7.03 Hz, 1 H) 24

3 HRMS m/z calcd for C₂₄H₂₈FN₄O₄ 455.2089 [M + H]⁺, found 455.2092. ¹HNMR (400 MHz, CD₃OD) δ ppm 2.21 (s, 3 H), 2.42 (s, 3 H), 2.75-2.96 (m, 5H), 3.02 (d, J = 6.25 Hz, 2 H), 3.34 (br. s., 1 H), 3.52 (br. s., 1 H),3.62 (s, 3 H), 3.76 (d, J = 11.72 Hz, 3 H), 6.83 (d, J = 6.64 Hz, 1 H),7.21 (d, J = 10.94 Hz, 1 H), 7.28 (s, 1 H), 7.66 (d, J = 7.03 Hz, 1 H),8.31 (d, J = 7.03 Hz, 1 H) 25

3 HRM m/z calcd for C₂₃H₂₆ClN₄O₃ 441.1688 [M + H]⁺, found 441.168. ¹HNMR (400 MHz, CD₃OD) δ ppm 1.99 (d, J = 13.67 Hz, 3 H), 2.26 (s, 3 H),2.33-2.72 (m, 1 H), 2.81-2.98 (m, 4 H), 2.99-3.20 (m, 2 H), 3.31- 3.42(m, 1 H), 3.42-3.71 (m, 2 H), 3.71-3.86 (m, 1 H), 4.19 (d, J = 12.89 Hz,1 H), 7.02 (d, J = 7.03 Hz, 1 H), 7.36-7.51 (m, 1 H), 7.58 (br. s., 1H), 7.69 (d, J = 6.25 Hz, 1 H), 7.78 (br. s., 1 H), 8.44-8.59 (m, 1 H)26

3 HRMS m/z calcd for C₂₄H₂₇ClFN₄O₃ 473.175 [M + H]⁺, found 473.1753. ¹HNMR (400 MHz, CD₃OD) δ ppm 0.99-1.13 (m, 3 H), 2.24 (s, 3 H), 2.26- 2.44(m, 2 H), 2.94-2.98 (m, 4 H), 3.06-3.14 (m, 2 H), 3.35-3.42 (m, 1 H),3.50-3.76 (m, 3 H), 3.75-3.86 (m, 1 H), 4.16-4.34 (m, 1 H), 6.98- 7.10(m, 1 H), 7.22-7.35 (m, 1 H), 7.59 (d, J = 1.95 Hz, 1 H), 7.70 (dd, J =7.42, 3.52 Hz, 1 H), 8.45-8.57 (m, 1 H). 27

3 HRMS m/z calcd for C₂₃H₂₆ClN₄O₃ 441.1688 [M + H]⁺, found 441.1692. ¹HNMR (400 MHz, CD₃OD) δ ppm 1.99 (d, J = 14.45 Hz, 3 H), 2.26 (s, 3 H),2.31-2.72 (m, 1 H), 2.82-2.95 (m, 3 H), 3.00-3.20 (m, 3 H), 3.35 (td, J= 11.72, 2.34 Hz, 1 H), 3.44-3.69 (m, 2 H), 3.78 (dd, J = 11.52, 2.54Hz, 1 H), 4.19 (d, J = 12.89 Hz, 1 H), 6.98 (d, J = 7.42 Hz, 1 H), 7.42(dd, J = 11.72, 7.81 Hz, 1 H), 7.55 (s, 1 H), 7.65-7.73 (m, 1 H), 7.77(br. s., 1 H), 8.43-8.52 (m, 1 H). 28

3 HRMS m/z calcd for C₂₂H₂₂ClF₂N₄O₄ 479.1292 [M + H]⁺, found 479.1290.¹H NMR (400 MHz, DMSO-d₆) δ ppm 2.79-2.84 (m, 3 H), 2.99-3.11 (m, 2 H),3.14-3.24 (m, 2 H), 3.30-3.70 (m, 5 H), 3.71-3.85 (m, 2 H), 7.08 (dd, J= 7.42, 1.95 Hz, 1 H), 7.65 (d, J = 8.20 Hz, 2 H), 7.75-7.79 (m, 1 H),8.58 (d, J = 7.42 Hz, 1 H), 8.72 (d, J = 4.30 Hz, 1 H). 29

3 HRMS m/z calcd for C₂₂H₂₂ClF₂N₄O₃ 463.1343 [M + H]⁺, found 463.1348.¹H NMR (400 MHz, DMSO-d₆) δ ppm 1.87 (d, J = 2.73 Hz, 3 H), 2.15-2.30(m, 1 H), 2.77 (d, J = 4.69 Hz, 3 H), 2.96-3.08 (m, 2 H), 3.15-4.15 (m,6H), 7.00-7.07 (m, 1 H), 7.61 (dd, J = 8.20, 2.73 Hz, 2 H), 7.70-7.74(m, 1 H), 8.54 (t, J = 6.84 Hz, 1 H), 8.68 (d, J = 4.69 Hz, 1 H). 30

3 HRM m/z calcd for C₂₂H₂₄ClF₂N₄O₃ 477.1500 [M + H]⁺, found 477.1503. ¹HNMR (400 MHz, DMSO-d₆) δ ppm 0.87 (t, J = 7.42 Hz, 3 H) 2.00-2.29 (m, 3H) 2.74-2.80 (m, 3 H) 2.99- 3.30 (m, 3 H) 3.50-3.80 (m, 4H), 4.05- 4.21(m, 1 H) 7.00-7.07 (m, 1 H) 7.61 (d, J = 7.81 Hz, 2 H) 7.72 (d, J = 1.56Hz, 1 H) 8.54 (t, J = 6.84 Hz, 1 H) 8.68 (d, J = 4.30 Hz, 1 H). 31

3 HRM m/z calcd for C₂₅H₃₁N₄O₃ 435.2391 [M + H]⁺, found 435.2395. ¹H NMR(400 MHz, CD₃OD) δ ppm 1.04 (dt, J = 12.11, 7.62 Hz, 3 H), 2.18- 2.42(m, 5 H), 2.45 (s, 3 H), 2.65- 2.70 (m, 1 H), 2.86 (dd, J = 13.09, 10.35Hz, 1 H), 2.95 (s, 3 H), 3.02- 3.20 (m, 2 H), 3.34-3.42 (m, 1 H),3.46-3.62 (m, 1 H), 3.67 (t, J = 13.87 Hz, 1 H), 3.81 (dd, J = 11.91,2.15 Hz, 1 H), 4.21 (d, J = 12.89 Hz, 1 H), 6.86 (d, J = 7.03 Hz, 1 H),7.31 (s, 1 H), 7.39-7.51 (m, 1 H), 7.65-7.76 (m, 1 H), 7.79 (br. s., 1H), 8.27-8.43 (m, 1 H). 32

3 HRM m/z calcd for C₂₃H₂₅F₂N₄O₃ 443.1886 [M + H]⁺, found 443.1889. ¹HNMR (400 MHz, DMSO-d₆) δ ppm 1.91 (d, J = 6.25 Hz, 3 H), 2.37 (s, 3 H),2.81 (d, J = 4.7 Hz, 3 H), 2.95- 3.07 (m, 2 H), 3.10-3.45 (m, 3 H),,3.50-4.50 (m, 4 H), 6.80-6.89 (m, 1 H), 7.35 (s, 1 H), 7.63 (dd, J =8.20, 3.12 Hz, 2 H), 8.37-8.46 (m, 1 H), 8.71 (d, J = 4.30 Hz, 1 H). 33

3 HRMS m/z calcd for C₂₅H₃₁N₄O₃ 435.2391 [M + H]⁺, found 435.2398. ¹HNMR (400 MHz, CD₃OD) δ ppm 1.22 (t, J = 7.23 Hz, 3 H), 1.98 (d, J =16.80 Hz, 3 H), 2.26 (s, 3 H), 2.42 (s, 3 H), 2.59-2.91 (m, 1 H),2.96-3.18 (m, 3 H), 3.31-3.53 (m, 4 H), 3.54- 3.68 (m, 1 H), 3.78 (d, J= 11.72 Hz, 1 H), 4.17 (d, J = 12.89 Hz, 1 H), 6.82 (d, J = 7.03 Hz, 1H), 7.28 (s, 1 H), 7.36-7.47 (m, 1 H), 7.63-7.72 (m, 1 H), 7.76 (br. s.,1 H), 8.33 (dd, J = 11.91, 7.23 Hz, 1 H) 34

4 HRMS m/z calcd for C₂₃H₂₅F2N₄O₄ 459.1836 [M + H]⁺, found 459.1838. ¹HNMR (400 MHz, CD₃OD) δ ppm 2.45 (s, 3 H), 2.53-2.69 (m, 1 H), 2.78- 2.99(m, 4 H), 3.01-3.18 (m, 2 H), 3.33-3.39 (m, 1 H), 3.50-3.62 (m, 1 H),3.65 (s, 3 H), 3.72-3.87 (m, 3 H), 6.86 (dd, J = 7.03, 1.56 Hz, 1 H),7.33 (s, 1 H), 7.59 (d, J = 8.20 Hz, 2 H), 8.40 (d, J = 7.03 Hz, 1 H).35

4 HRMS m/z calcd for C₂₄H₂₇F₂N₄O₃ 457.2046 [M + H]⁺, found 457.2043. ¹HNMR (400 MHz, DMSO-0d₆) δ ppm 0.60 (t, J = 7.42 Hz, 3 H), 1.76-2.04 (m,2 H), 2.06 (s, 3 H), 2.15-2.29 (m, 4 H), 2.35-2.47 (m, 0.5 H), 2.59-2.78 (m, 2 H), 2.99 (s, 3 H), 3.04-3.08 (s, 1 H), 3.25-3.46 (m, 0.5 H),3.65- 3.86 (m, 1 H), 6.52 (d, J = 6.64 Hz, 1 H), 7.03 (s, 1 H), 7.33 (d,J = 7.81 Hz, 2 H), 8.05-8.18 (m, 1 H), 8.36 (d, J = 4.69 Hz, 1 H) 36

4 MS (ESI) m/z 448.11 [M + H]⁺; MS (ESI) m/z 470.09 [M + Na]⁺. ¹H NMR(300 MHz, CDCl₃) δ ppm 2.29 (s, 3 H), 2.50-2.62 (m, 1 H), 2.80- 2.97 (m,1 H), 3.00 (d, J = 5.95 Hz, 2 H), 3.05 (d, J = 4.87 Hz, 3 H), 3.30- 3.41(m, 1 H), 3.46-3.56 (m, 1 H), 3.68 (s, 3 H), 3.70-3.95 (m, 3 H), 6.16-6.23 (m, 1 H), 6.98 (dd, J = 7.17, 1.64 Hz, 1 H), 7.33 (d, J = 7.83 Hz,1 H), 7.64 (d, J = 8.08 Hz, 1 H), 7.74 (s, 1 H), 8.00-8.01 (m, 1 H),8.38 (d, J = 7.21 Hz, 1 H) 37

5 MS (ESI) m/z 435.16 [M + H]⁺. ¹H NMR (300 MHz, CDCl₃) δ ppm 0.80-0.92(m, 1 H), 1.18-1.32 (m, 2 H), 1.45-1.60 (m, 1 H), 1.65-1.80 (m, 1 H),2.24-2.30 (m, 1 H), 2.32 (s, 3 H), 2.42 (s, 3 H), 2.57-2.81 (m, 3 H),3.03 (d, J = 4.87 Hz, 3 H), 3.61 (s, 3 H), 3.80-4.05 (m, 2 H), 6.23-6.30(m, 1 H), 6.70 (dd, J = 6.98, 1.35 Hz, 1 H), 7.34 (d, J = 7.90 Hz, 1 H),7.37 (s, 1 H), 7.59 (dd, J = 7.90, 1.44 Hz, 1 H), 7.69 (d, J = 1.11 Hz,1 H), 7.83 (d, J = 6.98 Hz, 1 H) 38

5 MS (ESI) m/z calcd for C₂₅H₃₁N₄O₂ 419.24 [M + H]⁺, found 419.18. ¹HNMR (300 MHz, CDCl₃) δ ppm 0.78-0.90 (m, 1 H), 1.18-1.30 (m, 2 H),1.47-1.54 (m, 1 H), 1.65-1.78 (m, 1 H), 1.83 and 2.02 (2 s, 3 H), 2.17-2.25 (m, 0.75 H), 2.33 (s, 3 H), 2.41- 2.44 (m, 3 H), 2.62-2.91 (m, 2.25H), 3.02-3.06 (m, 3 H), 3.35-3.50 (m, 1 H), 3.50-3.65 (m, 1 H),4.28-4.45 (m, 1 H), 6.15-6.22 (m, 1 H), 6.67- 6.75 (m, 1 H), 7.34-7.39(m, 2 H), 7.57-7.61 (m, 1 H), 7.68-7.72 (m, 1 H), 7.81-7.85 (m, 1 H) 39

5 MS (ESI) m/z 439.09 [M + H]⁺. ¹H NMR (300 MHz, CDCl₃) δ ppm 1.93 and2.04 (2 s, 3 H), 2.27 (s, 3 H), 2.29-2.35 (m, 0.5 H), 2.39 (s, 3 H),2.58-2.69 (m, 0.5 H), 2.77 (dd, J = 12.94, 10.69 Hz, 0.5 H), 2.93 (t, J= 5.62 Hz, 2 H), 3.01 (d, J = 4.73 Hz, 3 H), 3.12-3.22 (m, 0.5 H),3.30-3.55 (m, 3 H), 3.82-3.90 (m, 1 H), 4.31 (t, J = 12.13 Hz, 1 H),6.41-6.53 (m, 1 H), 7.30 (d, J = 7.87 Hz, 1 H), 7.36 (d, J = 7.05 Hz, 1H), 7.59 (t, J = 8.26 Hz, 1 H), 7.70 (s, 1 H), 8.15 (dd, J = 17.69, 5.14Hz, 1 H) 40

5 MS (ESI) m/z 435.12 [M + H]⁺; MS (ESI) m/z 457.10 [M + Na]⁺. ¹H NMR(300 MHz, CDCl₃) δ ppm 1.09-1.26 (m, 3 H), 1.75-2.04 (m, 3 H), 2.32 (s,3 H), 2.42 (s, 3 H), 2.98- 3.09 (m, 1 H), 3.03 (d, J = 4.76 Hz, 3 H),3.16-3.28 (m, 3 H), 3.40-3.77 (m, 3 H), 3.98 (br s, 1 H), 6.28 (d, J =4.00 Hz, 1 H), 6.72 (d, J = 6.73 Hz, 1 H), 7.35-7.39 (m, 2 H), 7.58 (d,J = 7.86 Hz, 1 H), 7.71 (s, 1 H), 7.96 (d, J = 6.99 Hz, 1 H) 41

5 MS (ESI) m/z 421.13 [M + H]⁺ ¹H NMR (300 MHz, CDCl₃) δ ppm 1.32-1.42(m, 1 H), 1.72-1.84 (m, 1 H), 2.31 (s, 3 H), 2.39-2.44 (m, 1 H), 2.43(s, 3 H), 2.77-2.85 (m, 1 H), 2.89 (d, J = 7.55 Hz, 2 H), 3.03 (d, J =4.88 Hz, 3 H), 3.12-3.48 (m, 3 H), 3.61 (d, J = 3.54 Hz, 3 H), 6.31 (brs., 1 H), 6.71 (dd, J = 7.00, 1.63 Hz, 1 H), 7.31- 7.38 (m, 2 H), 7.58(d, J = 7.79 Hz, 1 H), 7.70 (s, 1 H), 7.85 (d, J = 6.95 Hz, 1 H) 42

5 MS (ESI) m/z 419.09 [M + H]⁺; MS (ESI) m/z 441.06 [M + Na]⁺. ¹H NMR(300 MHz, CDCl₃) δ ppm 1.02-1.19 (m, 3 H), 1.45-1.59 (m, 1 H), 1.58-1.77(m, 1 H), 1.80, 1.86, 1.96 and 1.97 (4 s, 3 H), 2.02-2.16 (m, 0.5 H),2.33 (s, 3 H), 2.44 (s, 3 H), 2.53-2.67 (m, 0.5 H), 2.72-2.88 (m, 0.5H), 2.90-2.97 (m, 2 H), 2.98- 3.06 (m, 3 H), 3.27-3.35 (m, 0.5 H), 3.50(dd, J = 12.11, 7.70 Hz, 0.5 H), 3.72-3.80 (m, 0.5 H), 3.85-4.00 (m, 0.5H), 4.05-4.13 (m, 0.5 H), 6.22- 6.30 (m, 1 H), 6.69-6.76 (m, 1 H), 7.35(d, J = 7.92 Hz, 1 H), 7.40 (s, 1 H), 7.57-7.64 (m, 1 H), 7.70-7.73 (m,1 H), 7.81-7.89 (m, 1 H) 43

5 MS (ESI) m/z 435.11 [M + H]⁺, f; MS (ESI) m/z 457.09 [M + Na]⁺ ¹H NMR(300 MHz, CDCl₃) δ ppm 1.10 and 1.25 (2 d, J = 6.89 and 6.81 Hz, 3 H),1.88 and 2.04 (2 s, 3 H), 2.30 (s, 3 H), 2.41 and 2.42 (2 s, 3 H), 2.47-2.52 (m, 0.5 H), 2.84-3.16 (m, 5 H), 3.38-3.50 (m, 2 H), 3.61-3.74 (m,1.5 H), 4.16 (dd, J = 13.63, 2.70 Hz, 0.5 H), 4.44-4.51 (m, 0.5 H),6.33- 6.44 (m, 1 H), 6.65-6.71 (m, 1 H), 7.32-7.36 (m, 2 H), 7.56-7.62(m, 1 H), 7.68-7.71 (m, 1 H), 8.07 and 8.15 (2 d, J = 7.06 and 7.24 Hz,1 H) 44

5 MS (ESI) m/z 405.16 [M + H]⁺. ¹H NMR (300 MHz, CDCl₃) δ ppm 1.35-1.49(m, 1 H), 1.76-1.92 (m, 1 H), 1.84 and 1.92 (2 s, 3 H), 2.32 (d, J =2.07 Hz, 3 H), 2.36-2.55 (m, 1 H), 2.43 (s, 3 H), 2.79-2.98 (m, 3 H),3.03 (d, J = 4.88 Hz, 3 H), 3.20-3.33 (m, 2 H), 3.41-3.57 (m, 1 H), 6.33(br s, 1 H), 6.71-6.75 (m, 1 H), 7.35 (d, J = 7.85 Hz, 1 H), 7.37-7.41(m, 1 H), 7.59 (dt, J = 7.96, 1.84 Hz, 1 H), 7.69- 7.73 (m, 1 H), 7.86(dd, J = 6.96, 3.14 Hz, 1 H) ¹These compounds were analyzed byanalytical HPLC (UV, ELSD and MS) using a low pH method (mobile phase:A: H₂O with 0.05% TFA; B: CH₃CN with 0.05% TFA; Zorbax SB C18, Agilentreverse phase column; column size: 4.6 × 30 mm; particle size: 1.8 μm;4.5 min. run with a gradient of 5-95% B in A).

Example 45. Preparation of methyl4-((7-methyl-2-(2-methyl-4-(methylcarbamoyl)phenyl)imidazo[1,2-a]pyridin-3-yl)methyl)piperidine-1-carboxylate

Part A. Preparation of methyl 4-(1-ethoxyvinyl)-3-methylbenzoate

A mixture of methyl 4-bromo-3-methylbenzoate (5 g, 21.83 mmol),PdCl₂(dbpf) (0.356 g, 0.55 mmol) and tributyl(1-ethoxyvinyl)stannane(8.11 mL, 24.01 mmol) in 1,4-dioxane (20 mL) was heated at 150° C.during 15 min in a microwave reactor. The mixture was filtered on a padof diatomaceous earth, which was subsequently washed with EtOAc (100mL). Brine (60 mL) was added to the resulting mixture, and the phaseswere separated.

The aqueous phase was extracted with EtOAc (3×60 mL). The combinedorganic extracts were dried over magnesium sulfate, filtered, andconcentrated under reduced pressure. The residue was used withoutfurther purification in the next step. LCMS m/z 221.09 [M+H]⁺ (ESI).

Part B. Preparation of methyl 4-(2-bromoacetyl)-3-methylbenzoate

Methyl 4-(1-ethoxyvinyl)-3-methylbenzoate (4.81 g, 21.83 mmol) wasdissolved in THF (30 mL) and water (15.00 mL) at 22° C. and1-bromopyrrolidine-2,5-dione (3.89 g, 21.83 mmol) was added. Theresulting mixture was stirred at 22° C. for 15 min. EtOAc (70 mL) andwater (50 mL) were added to the resulting mixture, and the phases wereseparated. The aqueous phase was extracted with EtOAc (3×50 mL). Thecombined organic extracts were dried over magnesium sulfate, filtered,and concentrated under reduced pressure. The residue was purified byflash chromatography on silica gel, eluting with mixtures of EtOAc andheptane to afford methyl 4-(2-bromoacetyl)-3-methylbenzoate (4.51 g,76%) as a solid. ¹H NMR (400 MHz, CDCl₃) δ ppm 2.55 (s, 3H), 3.95 (s,3H), 4.41 (s, 2H), 7.68 (d, 1H), 7.89-8.00 (m, 2H)

Part C. Preparation of methyl3-methyl-4-(7-methylimidazo[1,2-a]pyridin-2-yl)benzoate

A mixture of 4-methylpyridin-2-amine (1.149 g, 10.63 mmol), methyl4-(2-bromoacetyl)-3-methylbenzoate (2.4 g, 8.85 mmol) and sodiumbicarbonate (1.487 g, 17.71 mmol) in ethanol (8 mL) was heated in amicrowave reaction to 120° C. during 10 min. After cooling to rt, theprecipitate was collected by filtration, washed with water and driedunder reduced pressure to afford methyl3-methyl-4-(7-methylimidazo[1,2-a]pyridin-2-yl)benzoate (1.49 g, 60%) asa solid. ¹H NMR (400 MHz, CD₃OD) δ ppm 2.43 (s, 3H), 2.56 (s, 3H), 3.92(s, 3H), 6.81 (d, J=6.64 Hz, 1H), 7.34 (s, 1H), 7.83-7.87 (m, 1H),7.87-7.92 (m, 1H), 7.95 (s, 1H), 7.99 (s, 1H), 8.33 (d, J=7.03 Hz, 1H)LCMS m/z 281.02 [M+H]⁺ (ESI).

Part D. Preparation ofN,3-dimethyl-4-(7-methylimidazo[1,2-a]pyridin-2-yl)benzamide

A mixture of methyl3-methyl-4-(7-methylimidazo[1,2-a]pyridin-2-yl)benzoate (1.47 g, 5.24mmol), 40% water solution of methanamine (10 ml, 116.17 mmol) was heatedin a microwave reactor to 105° C. during 10 min. EtOAc (50 mL) was addedto the resulting mixture, the mixture was were filtered, and the twophases separated. The aqueous phase was extracted with EtOAc (3×50 mL).The combined organic phases were dried over magnesium sulfate, filtered,and concentrated under reduced pressure. The residue was purified byflash chromatography on silica gel, eluting with mixtures of EtOAc andmethanol, to affordN,3-dimethyl-4-(7-methylimidazo[1,2-a]pyridin-2-yl)benzamide (1.1 g,75%) as a solid. 1H NMR (400 MHz, CD₃OD) δ ppm 2.43 (s, 3H), 2.56 (s,3H), 2.94 (s, 3H), 6.80 (dd, J=7.03, 1.56 Hz, 1H), 7.34 (s, 1H),7.67-7.74 (m, 1H), 7.76 (s, 1H), 7.82 (d, J=7.81 Hz, 1H), 7.97 (s, 1H),8.33 (d, J=7.03 Hz, 1H) LCMS m/z 280.10 [M+H]⁺ (ESI).

Part E. Preparation of4-(3-bromo-7-methylimidazo[1,2-a]pyridin-2-yl)-N,3-dimethylbenzamide

A solution of dibromine (0.051 mL, 0.99 mmol) in ethanol (1 mL) wasadded dropwise to a stirred solution ofN,3-dimethyl-4-(7-methylimidazo[1,2-a]pyridin-2-yl)benzamide (184 mg,0.66 mmol) in ethanol (1.000 mL) at room temperature. The resultingmixture was stirred 90 min, concentrated under reduced pressure and theresidue was suspended in water. The aqueous mixture was made basic usingNaHCO₃ and extracted with CH₂Cl₂. The organic phase was dried overMgSO₄, concentrated under reduced pressure and the residue was purifiedby silica gel flash chromatography (gradient 7-60% EtOAc in heptane) togive4-(3-bromo-7-methylimidazo[1,2-a]pyridin-2-yl)-N,3-dimethylbenzamide(210 mg, 89%) as a solid. LCMS m/z 357.98, 359.97 [M+H]⁺ (ESI).

Part F. Preparation of tert-butyl4-((7-methyl-2-(2-methyl-4-(methylcarbamoyl)phenyl)imidazo[1,2-a]pyridin-3-yl)methylene)piperidine-1-carboxylate

To a mixture of PdCl₂(dbpf) (60.0 mg, 0.09 mmol) and tetrabutylammoniumchloride (25.6 mg, 0.09 mmol) under N₂,4-(3-bromo-7-methylimidazo[1,2-a]pyridin-2-yl)-N,3-dimethylbenzamide(330 mg, 0.92 mmol) and tert-butyl 4-methylenepiperidine-1-carboxylate(545 mg, 2.76 mmol) in DMA (10 mL) were added, and the resulting mixturewas heated in a microwave reactor at 120° C. for 45 min. The resultingmixture was filtered over diatomaceous earth. The filtrate wasconcentrated under reduced pressure, then EtOAc (20 mL) and water (20mL) were added. The phases were separated and the aqueous phase wasextracted with EtOAc (3×20 mL). The combined organic phases were driedover magnesium sulfate, filtered, and concentrated under reducedpressure. The residue was used in the next step without furtherpurification. LCMS m/z 475.30 [M+H](ESI).

Part G. Preparation of tert-butyl4-((7-methyl-2-(2-methyl-4-(methylcarbamoyl)phenyl)imidazo[1,2-a]pyridin-3-yl)methyl)piperidine-1-carboxylate

The pressure of the H-cube was set to 60 bar and the temperature to 60°C. with a 30 mm cartridge containing 10% Pd/C. A solution of tert-butyl4-((7-methyl-2-(2-methyl-4-(methylcarbamoyl)phenyl)imidazo[1,2-a]pyridin-3-yl)methylene)piperidine-1-carboxylatein ethanol (5 mL) and ethyl acetate (5 mL) was pumped through the H-Cubeat a rate of mL/min. After completion, the fractions were concentratedand the residue was used in the next step without further purification.MS m/z 477.30 [M+H]⁺ (ESI).

Part H. Preparation of methyl4-((7-methyl-2-(2-methyl-4-(methylcarbamoyl)phenyl)imidazo[1,2-a]pyridin-3-yl)methyl)piperidine-1-carboxylate

A mixture of tert-butyl4-((7-methyl-2-(2-methyl-4-(methylcarbamoyl)phenyl)imidazo[1,2-a]pyridin-3-yl)methyl)piperidine-1-carboxylatecompound containingN,3-dimethyl-4-(7-methylimidazo[1,2-a]pyridin-2-yl)benzamide (180 mg,1:1 mixture) and HCl (0.022 mL, 0.71 mmol) in MeOH (2 mL) was heated at70° C. for 1 hr. The resulting mixture was cooled to rt, concentratedunder reduced pressure, and the residue was dissolved in dichloromethane(2.000 mL). Methyl chloroformate (0.028 mL, 0.36 mmol) and DIPEA (0.250mL, 1.43 mmol) were added to the solution, and the resulting mixture wasstirred at 0° C. for 1 hr. After concentration under reduced pressure,the residue was purified on preparative HPLC MS (Mobile phase: 30-50% B;A: H₂O with 10 mM NH₄CO₃ and 0.375% NH₄OH v/v, B: CH₃CN, 25 min run) onXBridge Prep C18 OBD, 30×150 mm, 5 μm, Waters reverse phase column, toafford methyl4-((7-methyl-2-(2-methyl-4-(methylcarbamoyl)phenyl)imidazo[1,2-a]pyridin-3-yl)methyl)piperidine-1-carboxylate(11.00 mg, 10.63%) as a solid. ¹H NMR (400 MHz, METHANOL-d₄) δ ppm0.81-1.03 (m, 2H), 1.49 (d, J=12.50 Hz, 2H), 1.71-1.89 (m, 1H), 2.30 (s,3H), 2.45 (s, 3H), 2.66 (br. s., 2H), 2.84 (d, J=7.42 Hz, 2H), 2.94 (s,3H), 3.61 (s, 3H), 3.92 (d, J=5.08 Hz, 2H), 6.88 (dd, J=7.23, 1.76 Hz,1H), 7.32 (s, 1H), 7.40 (d, J=7.81 Hz, 1H), 7.70 (dd, J=7.81, 1.95 Hz,1H), 7.79 (s, 1H), 8.25 (d, J=7.03 Hz, 1H). HRMS m/z calcd forC₂₅H₃₁N₄O₃ 435.2391 [M+H]⁺, found 435.2405.

Example 46. Preparation of4-(3-((1-acetylpiperidin-4-yl)methyl)-7-methylimidazo[1,2-a]pyridin-2-yl)-N,3-dimethylbenzamide

A mixture of tert-butyl4-((7-methyl-2-(2-methyl-4-(methylcarbamoyl)phenyl)imidazo[1,2-a]pyridin-3-yl)methyl)piperidine-1-carboxylatecompound containingN,3-dimethyl-4-(7-methylimidazo[1,2-a]pyridin-2-yl)benzamide (200 mg,1:1 mixture) and hydrogen chloride (32.9 mg, 0.90 mmol) in MeOH (3.0 mL)was heated at 70° C. for 1 hr. After concentration, the residue wasdissolved in CH₂Cl₂ (3.00 mL), followed by addition of pyridine (143 mg,1.80 mmol) and acetic anhydride (30.7 mg, 0.30 mmol). The resultingmixture was stirred at 0° C. for 1 hr. After concentration, the crudewas purified by flash chromatography on silica gel, eluting withmixtures of ethyl acetate and methanol, and then purification bypreparative HPLC (UV collection) using a low pH method (Mobile phase:A-H2O with 0.05% TFA; B-CH3CN, on Luna C18, 50×250 mm, 15 um Phenomenexreverse phase column), 20 min run with a gradient of 10-20% B in A thenpurification by HPLC (MS collection) using a high pH method (Mobilephase: A-H2O with 10 mM NH5CO3 and 0.375% NH4OH v/v; B-MeOH, onGemini-NX C18 110A, Axia, 30×150 mm, 5 um Phenomenex reverse phasecolumn), 19.5 min. run with a gradient of 40-60% B in A thenpurification with a MettlerToledo Minigram Supercritical FluidChromatography instrument using the following conditions: Cyano Column,10.0×250 mm, 6 μm particle size, 10.0 mL/min, mobile phase: 25% EtOHwith 0.1% DMEA, supercritical CO₂, regulator set to 100 Bar, columntemperature set to 35° C., UV 215 nm, providing4-(3-((1-acetylpiperidin-4-yl)methyl)-7-methylimidazo[1,2-a]pyridin-2-yl)-N,3-dimethylbenzamide(7.00 mg, 16.68%) as a solid. ¹H NMR (400 MHz, methanol-d₄). δ ppm0.82-1.09 (m, 2H), 1.35 (d, J=10.55 Hz, 1H), 1.46-1.66 (m, 2H), 1.87(ddd, J=15.14, 7.52, 4.30 Hz, 1H), 2.00 (s, 3H), 2.30 (s, 3H), 2.40-2.52(m, 4H), 2.83-3.02 (m, 5H), 3.74 (br. s., 1H), 4.33 (d, J=13.28 Hz, 1H),6.89 (dd, J=7.03, 1.56 Hz, 1H), 7.33 (s, 1H), 7.41 (d, J=7.81 Hz, 1H),7.71 (d, J=8.20 Hz, 1H), 7.79 (s, 1H), 8.27 (d, J=7.03 Hz, 1H); HRMS m/zcalcd for C₂₅H₃₁N₄O₂ 419.2442 [M+H]⁺, found 419.2437.

Example 47. Preparation of(R)-4-(3-((4-acetylmorpholin-2-yl)methyl)-7-methylimidazo[1,2-a]pyridin-2-yl)-N,3-dimethylbenzamide

¹H NMR (400 MHz, CD₃OD) δ ppm 2.02 (2 s, 3H), 2.30 (s, 3H), 2.38 (dd,J=13.09, 10.74 Hz, 1H), 2.45 (s, 3H), 2.60-2.74 (m, 1H), 2.84-2.95 (m,3H), 3.03-3.10 (m, 2H), 3.10-3.22 (m, 1H), 3.59-3.70 (m, 2H), 3.81 (dd,J=11.91, 3.32 Hz, 1H), 4.21 (d, J=13.28 Hz, 1H), 6.86 (d, J=7.03 Hz,1H), 7.31 (s, 1H), 7.40-7.51 (m, 1H), 7.66-7.76 (m, 1H), 7.79 (br. s.,1H), 8.37 (dd, J=12.11, 7.03 Hz, 1H). HRMS m/z calcd for C₂₄H₂₉N₄O₂421.2234 [M+H]⁺, found 421.2237, R_(t) 0.993 min. Applicants synthesizedthis compound using a relatively complicated process. If applicants wereto re-synthesize this compound, they would instead use a process similarto that used to prepare the compound in Example 20.

Example 48. Biological Evaluation of Compounds as Antagonists at HumanP2X3 Receptor In Vitro

The antagonist properties of compounds in the present invention wereassayed as inhibitors of intracellular calcium increase induced byactivation of hP2X3 (human Purinergic P2X receptors subtype 3, accessionnumber AB016608 for clone A and accession number NM_002559 for clone B),expressed in RLE cells (rat liver endothelium, ATCC. The RLE/hP2X3 cellswere grown in William's medium 1× (Gibco, 12551-032), supplemented with10% Fetal bovine serum (Wisent, 090850), 2 mM L-Glutamine (Wisent,609-065-EL), and 600 μg/mL Geneticin G-418 (Wisent, 61234) in ahumidified incubator (5% CO₂ and 37° C.).

Fluo-4 assay on FDSS7000 (Hamamatsu) was performed using cryopreservedRLE cells stably expressing hP2X3 plated in 384 well plates, 24 hrbefore the experiment at a density appropriate for obtaining the desiredfinal confluence. After processing the cell plates with Fluo-4 andperforming a subsequent incubation followed by washing steps, a doubleaddition was carried out. The first addition included the test compoundsdiluted in HBSS buffer containing 2 mM CaCl₂ preincubated for 20 minbefore a second addition. The second addition included 2 uM of ATP.Calcium mobilization was measured with the FDSS7000 over a time lapse of3 min, and fluorescent counts were exported for analysis. This resultedin a pIC₅₀, which was calculated in Activity base with ExcelFit. Hillcoefficients and % inhibitions can also be determined.

IC₅₀'s obtained using the above methods are shown in Table 2.

TABLE 2 IC₅₀'s Observed for the Compounds of Examples 15-47 Ex HumanP2X3 IC₅₀ (μM) 15 0.003 16 0.003 17 0.006 18 0.007 19 0.158 20 0.004 210.007 22 0.045 23 0.018 24 0.010 25 0.007 26 0.011 27 0.111 28 0.006 290.005 30 0.006 31 0.005 32 0.003 33 0.007 34 0.030 35 0.016 36 0.114 370.011 38 0.010 39 0.010 40 0.074 41 0.084 42 0.059 43 0.008 44 0.103 450.026 46 0.066 47 0.087

Example 49. Biological Evaluation of Example 15 in an In Vivo Model ofInflammatory Pain

One of the compounds of the present invention was evaluated in an invivo model of inflammatory pain. Oral dosing of Example 15(0.3-1-3-10-20 μmol/kg) produced a free plasma concentration dependentreversal of both heat and mechanical hyperalgesia endpoints in the ratFCA 96 hr paw model of inflammatory pain. The potency (EC50 free plasmaconcentration) of example 15 was 18 nM and 87 nM respectively inreversing the heat and mechanical hyperalgesia. The results are shown inFIGS. 1 and 2.

Methods for Characterization of Analgesic Effects of Example 15 in theFCA 96 hr Rat Model of Inflammatory Pain:

Male Sprague-Dawley rats (Charles River, St-Constant, Qc, CAN) weighing200-225 g were utilized for the animal experiment studies. The animalswere group-housed in polycarbonate, ventilated cages (filter top) in acontrolled environment room (12-h light/dark cycle, 20.5-23.5° C.,relative humidity: 40-70%) with food (14% Protein Rodent MaintenanceDiet, Harlan Teklad, Madisson, Wisc., USA) and water ad libidum.

Inflammation was induced by injection of a single 40 μl Freund'scomplete adjuvant (FCA) intra-plantar injection into the rat's lefthindpaw. All experiments were conducted 96 h after FCA administration.Twenty-four hours prior to behavioral testing animals were brought tothe laboratory for acclimatization to the new environment. Animals weresacrificed immediately after data acquisition.

Heat hyperalgesia (Plantar Test) was assessed using a Paw ThermalStimulator to apply a controlled heat to the plantar surface of theaffected paw. Rats were placed in individual plexiglass boxes withholes, on the glass surface of the device which was maintained at 30° C.They were allowed to habituate to their boxes for 15-30 minutes. Amovable arm containing the heat source, with an angled mirror, was usedto place the heat source directly under the injured paw withoutdisturbing the animal. The heat source and timer were then turned onsimultaneously; when the animal withdrew the hind paw the number ofseconds was recorded. Each animal was tested twice, 5 minutes apart, toavoid sensitization, and the average value was taken. To avoid anytissue damage, a cut-off time of 20 s was used.

Mechanical hyperalgesia was assessed using the Ugo Basile analgesy meter(Ugo Basile, Comerio, Italy). Animals were gently restrained, and asteadily increasing pressure was applied to the dorsal surface of a hindpaw via a probe with a dome-shaped tip (diameter of 1 mm). The pressurerequired to elicit paw withdrawal was determined. An assay cut off wasset at 295 g. Two trials were conducted with 5 min intervals betweeneach trial. Paw withdrawal thresholds were calculated as the mean of thetwo values. Animals were randomised and allocated to treatment groups toachieve a minimum statistical power of 80%. In all cases theexperimenter was blind to the treatment received.

Satellite animals were used to monitor the free plasma concentration ofexample 15 at the doses and time points used in the nociceptive tests.Free plasma EC50 values in behavioral studies were determined from4-point concentration-response curves using non-linear regression and asigmoidal variable slope logistic model (Prism 4.03, GraphPad SoftwareInc., USA).

Unless otherwise indicated, the following definitions are to be usedwhen reading this patent:

The chemical nomenclature used in this patent generally follows theexamples and rules stated in Nomenclature of Organic Chemistry, SectionsA, B, C, D, E, F, and H, Pergamon Press, Oxford, 1979.

The modifier “C_(m)-C_(n)” means that the modified group contains from mto n carbon atoms. For example, the term “C₁-C₆-alkyl” means an alkylgroup containing from 1 to 6 carbon atoms.

The term “alkyl” means a straight or branched chain alkane (hydrocarbon)radical. Examples of alkyl groups include, for example, methyl, ethyl,propyl, butyl pentyl, and hexyl.

The term “cycloalkyl” refers to a fully saturated cyclic hydrocarbongroup.

Examples of cycloalkyls include, for example, cyclopropyl, cyclobutyl,cyclopentyl, and cyclohexyl.

The term “halogen” means chlorine, bromine, fluorine, or iodine.

The term “alkoxy” means —O-alkyl. Examples of alkoxys include, forexample, methoxy, ethoxy, propoxy, and butoxy.

The term “optionally substituted” means that the modified group,structure, or molecule may be either: (1) substituted with a substituentat one or more substitutable positions, or (2) not substituted.

The term “pharmaceutically acceptable” is used to characterize a moiety(e.g., a salt, dosage form, carrier, diluent, or excipient) as beingappropriate for use in accordance with sound medical judgment. Ingeneral, a pharmaceutically acceptable moiety has one or more benefitsthat outweigh any deleterious effect that the moiety may have.Deleterious effects may include, for example, excessive toxicity,irritation, allergic response, and other problems and complications.

“Ac” means acetyl.

“AcOH” means acetic acid.

“AIBN” means azobisisobutylonitrile.

“atm” means atmosphere.

“boc” means tert-butyl carbonyl.

“Bu” means butyl.

“d” means doublet.

“DCM” means dichloromethane.

“dd” means doublet of doublet.

“ddd” means doublet of doublet of doublet.

“DIPEA” means diisopropylethylamine.

“DMA” means dimethylacetamide.

“DMEA” means dimethylethylamine.

“DMF” means N,N-dimethyl formamide.

“DMSO-d₆” means dimethylsulfoxide-d₆.

“DMT-MM” means 4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholiniumchloride.

“ESI” means electrospray ionization.

“Et” means ethyl.

“Et₂O” means diethyl ether.

“Et₃N” means triethylamine.

“EtOAc” means ethyl acetate.

“EtOH” means ethanol.

“Ex” means example.

“g” means gram.

“hr” means hour or hours.

“¹H NMR” means proton nuclear magnetic resonance.

“HATU” means O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate.

“HOBT” means 1-hydroxybenzotriazole.

“HPLC” means high-performance liquid chromatography.

“HRMS” means high-resolution mass spectrometry.

“L” means liter.

“LCMS” means liquid chromatography/mass spectroscopy.

“m” means multiplet.

“M” means molar.

“mL” means milliliter.

“Me” means methyl.

“MeCN” means acetonitrile.

“MeOH” means methanol.

“mg” means milligram.

“MHz” means megahertz.

“min” means minute or minutes.

“mmol” means millimole.

“mol” means mole.

“MS” means mass spectrometry.

“MTBE” means methyl tert-butyl ether.

“N” means normal.

“NBS” means N-bromosuccinimide.

“Pd(OH)₂” means Palladium hydroxide.

“Ph” means phenyl.

“ppm” means parts per million.

“Pr” means propyl.

“q” means quartet.

“qt” means quintet.

“R_(t)” means retention time (HPLC).

“s” means singlet.

“SFC” means supercritical-fluid chromatography.

“t” means triplet.

“TFA” means trifluoroacetic acid.

“THF” means tetrahydrofuran.

“TLC” means thin layer chromatography.

“TMEDA” means N,N,N′,N′-tetramethyl-1,2-ethylenediamine.

“UV” means ultraviolet.

“v/v” means volume per unit volume.

“vol” means volume.

References made in the singular may also include the plural. Forexample, “a” and “an” may refer to either one or more than one.

The words “comprise,” “comprises,” and “comprising” in this patent(including the claims) are to be interpreted inclusively rather thanexclusively. This interpretation is intended to be the same as theinterpretation that these words are given under United States patentlaw.

The above description of illustrative embodiments is intended only toacquaint others skilled in the art with the invention, its principles,and its practical application so that others skilled in the art mayadapt and apply the invention in its numerous forms, as they may be bestsuited to the requirements of a particular use. This invention,therefore, is not limited to the above embodiments, and may be variouslymodified.

We claim:
 1. A method for treating cough in an animal, wherein themethod comprises administering to the animal a therapeutically effectiveamount of a compound of Formula I or a salt thereof, wherein Formula Iis:

R¹ is selected from the group consisting of cyano, halogen, methyl, andethyl; R² is selected from the group consisting of hydrogen, halogen,methyl, and ethyl; R³ is selected from the group consisting of halogen,methyl, and ethyl; R⁴ is selected from the group consisting of hydrogen,halogen, methyl, ethyl, and methoxy; as to R⁵ and R⁶; R⁵ and R⁶ areindependently selected from the group consisting of hydrogen,C₁-C₆-alkyl, and hydroxy-C₁-C₆-alkyl; or R⁵ and R⁶, together with thenitrogen to which they are both attached, form a 5- or 6-memberheterocycloalkyl, wherein: the heterocycloalkyl is optionallysubstituted with one or more substituents independently selected fromthe group consisting of halogen, hydroxyl, and C₁-C₄-alkyl; R⁷ and R⁸are independently selected from the group consisting of hydrogen andC1-C₄-alkyl; R⁹ is selected from the group consisting of C₁-C₆-alkyl,C₃-C₆-cycloalkyl, C₁-C₆-alkyl-C₃-C₆-cycloalkyl, halo-C₁-C₆-alkyl,C₁-C₆-alkoxy, halo-C₁-C₆-alkoxy, and C₁-C₆-alkoxy-C₁-C₆-alkyl; and X isselected from a bond, CH₂, and O.
 2. The method of claim 1, wherein R¹is methyl and R² is hydrogen.
 3. The method of claim 1, wherein R³ andR⁴ are fluoro.
 4. The method of claim 1, wherein X is O.
 5. The methodof claim 1, wherein the compound corresponds in structure to:

and R⁴ is selected from the group consisting of halogen, methyl, andethyl.
 6. The method of claim 1, wherein R⁵ is hydrogen and R⁶ isC₁-C₆-alkyl.
 7. The method of claim 1, wherein R⁶ is methyl.
 8. Themethod of claim 1, wherein R⁷ and R⁸ are hydrogen.
 9. The method ofclaim 1, wherein R⁹ is C₁-C₆-alkoxy.
 10. The method of claim 1, whereinR⁹ is methoxy.
 11. The method of claim 1, wherein the compoundcorresponds in structure to:


12. The method of claim 1, wherein the compound corresponds in structureto:


13. The method of claim 1, wherein the compound corresponds in structureto:


14. The method of claim 1, wherein the compound corresponds in structureto:


15. The method of claim 1, wherein the animal is a mammal.
 16. Themethod of claim 15, wherein the mammal is a human.
 17. The method ofclaim 12, for treating cough in a human in need of such treatment. 18.The method of claim 13, for treating cough in a human in need of suchtreatment.
 19. The method of claim 14, for treating cough in a human inneed of such treatment.